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05 April 2022, Volume 49 Issue 4
Biotechnology
Bioinformatics Analysis and Expression of Elovl1 Gene in Muskrat Scent Gland
LEI Xing, LI Tianfeng, LI Jiahao, MAN Xin, BAI Suying
2022, 49(4):  1201-1212.  doi:10.16431/j.cnki.1671-7236.2022.04.001
Abstract ( 305 )   PDF (2315KB) ( 108 )  
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【Objective】 This study was aimed to determine the nucleotide sequence of elongase of verylong chain fatty acid 1 (Elovl1) gene in muskrat and analyze its bioinformatics,and explore its role in the development and secretion of muskrat scent gland.【Method】 The total RNA was extracted from adult male muskrat’s fragrant gland by TRIzol method,the sequence of Elovl1 gene in muskrat scent gland was obtained by transcriptome sequencing.The CDS sequence of Elovl1 gene and its multiple transcripts of different species of rodents were downloaded in GenBank database,and phylogenetic tree was constructed.The physicochemical property and structure of Elovl1 gene encoded protein were predicted and analyzed using online tools.The transcription level of Elovl1 gene was analyzed in scent gland tissue during the developmental period of adult male muskrat by Real-time quantitative PCR.【Result】 There were two transcripts of Elovl1 gene in muskrat scent gland,which could encode two different proteins,with lengths of 313 and 279 amino acids,respectively.Both transcripts of Elovl1 gene were expressed in muskrat scent gland from the end of February to the end of September,and the overall expression level showed a trend of up-regulation first and then down-regulation.The two proteins encoded by Elovl1 gene were bothstable lipophilic and non-secreted proteins,with 7 transmembrane helix regions,and their subcellular locations were mainly in the endoplasmic reticulum.In rodents,the genetic evolution of Elovl1 gene was basically the same as that of species.Circetidae and Muridae were closely related,Ondatra zibethicus and Arvicola amphibius were closely related.The different transcripts of Elovl1 gene of the same species were relatively conserved,both grouped by species and generally had higher confidence values,with the exception of Marmota.【Conclusion】 The expression pattern of Elovl1 gene was consistent with the development,atrophy and secretion period of muskrat scent gland,suggesting that Elovl 1 was related to muskrat scent secretion,and should be involved in the development of scent gland cells.
Analysis of Promoter Activity and Screening of Transcription Regulatory Elements of CCL19 Gene in Sheep
ZHAI Zhe, CHEN Si, WU Yanru, WANG Xuemei, LI Chongrui, LIU Zhiyong, CHEN Qiaoling, WANG Fengyang, DU Li, LI Chang, JIN Ningyi
2022, 49(4):  1213-1222.  doi:10.16431/j.cnki.1671-7236.2022.04.002
Abstract ( 209 )   PDF (3663KB) ( 62 )  
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【Objective】 The objective of this study was to identify the core promoter region and key transcription factors of sheep C-C motif chemokine ligand 19(CCL19) gene,and explore the transcription regulation mechanism of CCL19 gene.【Method】 Using sheep genomic DNA as template,the 5'-flanking sequence 1 000 bp of CCL19 gene was amplified by PCR.Moreover,seven active region sequences of CCL19 gene promoter were amplified and connected to the pGL3-Basic promoter vector.The recombined plasmid and pRL-TK plasmid were co-transfected into 293T cells,and the activity of relative luciferin enzymes was measured through dual-luciferase detection system.Transcription binding factor sites(TFBSs)prediction was made in the CCL19 gene promoter region by bioinformational method.The luciferase reporter vectors with TFBSs deletion were constructed using site mutation technology and co-transfected into 293T cells,the relative fluorescence activity of these deletion plasmids were analyzed.【Result】 The results showed that seven different length of CCL19 gene promoter fragments were successfully amplified and their dual-luciferase reporting vector (pGL3-P,pGL3-P1,pGL3-P2,pGL3-P3,pGL3-P4,pGL3-P5 and pGL3-P6) were constructed.The active detection of different length promoter fragments found that -256/-186 bp was the core promoter region of CCL19 gene,indicating that this region played an important role in the transcription regulation of CCL19 gene.Bio-information analysis of the CCL19 gene promoter region sequence found that there were 5 TFBSs in this region,including POU5F1(-201/-189 bp),ZBTB26(-228/-217 bp),FOXI1(-239/-228 bp),GLI2(-255/-243 bp) and SP2(-219/-211 bp),and the luciferase reporter vectors with TFBSs deletion were constructed.Dual-luciferase report results showed that the deletion of POU5F1 binding site extremely significantly reduced the transcriptional activity of CCL19 gene in sheep (P<0.01),and FOXI1,ZBTB26 and SP2 extremely significantly enhanced the transcriptional activity of CCL19 gene in sheep (P<0.01).【Conclusion】 This study indicated that the luciferase reporter vector of CCL19 gene promoter was successfully constructed and the core region of CCL19 gene promoter was determined.TFBSs of POU5F1 might be the transcriptional regulation site of CCL19 gene.This result could provide the theoretical basis for further exploring the function of CCL19 gene in lymphocyte migration,innate and adaptive immunity of sheep.
Study on Expression Characteristics of Myofiber Types and Energy Metabolism Related Genes in Pigs
ZHANG Yanwei, CAI Chunbo, SUN Di, AN Jiaqi, HUANG Xiaoyu, NIU Jin, CUI Zixu, GAO Pengfei, GUO Xiaohong, LI Bugao, CAO Guoqing
2022, 49(4):  1223-1232.  doi:10.16431/j.cnki.1671-7236.2022.04.003
Abstract ( 264 )   PDF (3613KB) ( 76 )  
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【Objective】 The aim of this study was to investigate the expression characteristics of myofiber types and energy metabolism related genes in Mashen pigs (MS),Jinfen White pigs (JFW) and Duroc×Landrace×Large (DLY) pigs.【Method】Six-month-old MS,JFW and DLY pigs were slaughtered,and the longissimus dorsi muscle (LD) and extensor digitorum longus muscle (EDL) were collected.HE staining was used to detect the diameter,density and cross-sectional area of myofiber.The expressions of Ⅰ,Ⅱa,Ⅱb and Ⅱx type myofiber marker genes (MYH7,MYH2,MYH4 and MYH1),mitochondrial activity related genes (ATP5A1,PGC1-α and PPARβ),electron transport chain related genes (UQCRC2,SDHA and NDUFA9) and glycolysis related genes (LDHA,LDHB and GK) were detected by Real-time quantitative PCR.【Result】 The diameter and cross-sectional area of longissimus dorsi muscle in MS pigs were significantly lower than those in JFW and DLY pigs,while the density was significantly higher than those in JFW and DLY pigs (P<0.05).The diameter and cross-sectional area of extensor digitorum longus muscle in MS and JFW pigs were significantly lower than those in DLY pigs,while the density was significantly higher than those in DLY pigs (P<0.05).The expression of MYH7 gene in longissimus dorsi muscle was the highest in MS pigs and the lowest in JFW pigs.The expression of MYH7 gene in extensor digitorum longus muscle of MS and JFW pigs were significantly higher than those of DLY pigs (P<0.05).The expression of MYH2 gene in longissimus dorsi muscle and extensor digitorum longus muscle of MS pigs were significantly higher than those of JFW and DLY pigs,and the expression of extensor digitorum longus muscle in JFW pigs was significantly higher than that in DLY pigs (P<0.05).The expression of MYH4 gene in longissimus dorsi muscle of MS pigs was significantly lower than those of JFW and DLY pigs,and the expression of extensor digitorum longus muscle in MS and JFW pigs were significantly lower than those in DLY pigs (P<0.05).The expression of ATP5A1 and PGC1-α genes in longissimus dorsi muscle of MS pigs were significantly lower than those of JFW and DLY pigs,and the expression of PGC1-α and PPARβ genes in JFW pigs were significantly lower than those in DLY pigs (P<0.05).The expression of ATP5A1,PGC1-α and PPARβ genes in extensor digitorum longus muscle of MS and JFW pigs were significantly lower than those of DLY pigs (P<0.05).The expression of UQCRC2,SDHA and NDUFA9 genes in longissimus dorsi muscle of JFW pigs were significantly lower than those of DLY and MS pigs (P<0.05).The expression of UQCRC2,SDHA and NDUFA9 genes in extensor digitorum longus muscle of JFW and MS pigs were significantly lower than those of DLY pigs,and the expression of UQCRC2 and SDHA genes in MS pigs were significantly lower than those in JFW pigs (P<0.05).The expression of LDHA,LDHB and GK genes in longissimus dorsi muscle and extensor digitorum longus muscle of JFW and MS pigs were significantly lower than those of DLY pigs (P<0.05).【Conclusion】 The diameter and cross-sectional area of skeletal muscle in MS pigs were smaller,and the expression of Ⅰ and Ⅱa myofiber marker genes was higher.The expression of Ⅱb myofiber marker gene,oxidative phosphorylation and glycolysis related genes in DLY pigs was higher.
Construction of Eukaryotic Expression Vector of Bovine Pregnancy-associated Glycoprotein 19 and Its Bioinformatics Analysis
DONG Yuan, YU Hongxu, CHEN Guoliang, BAO Dongbo, CUI Jiarui, LIU Lei, WANG Huiyan
2022, 49(4):  1233-1243.  doi:10.16431/j.cnki.1671-7236.2022.04.004
Abstract ( 220 )   PDF (2773KB) ( 94 )  
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【Objective】 The aim of this study was to amplify the bovine pregnancy-associated glycoprotein 19 (BoPAG19) gene,construct a eukaryotic expression vector,and detect its expression in HEK-293F cells.【Method】 BoPAG19 gene was synthesized in vitro according to the sequence of BoPAG19 gene (GenBank accession No.:NM_176628),and the target gene was amplified by PCR.After double enzyme digestion,the recombinant plasmid pcMV3-BoPAG19 was constructed and transfected into HEK-293F cells by Lipofectamine® 2000.BoPAG19 protein was expressed in the supernatant of cell culture and determined by SDS-PAGE,and then was purified by affinity chromatography.The hydrophobicity,transmembrane region,signal peptide,B cell antigen,secondary structure,tertiary structure and protein interaction of BoPAG19 protein were analyzed using bioinformatics softwares.【Result】 The recombinant vector pcMV3-BoPAG19 was successfully constructed.The length of BoPAG19 gene was about 1 200 bp,and the expression protein was about 60 ku.Bioinformatics analysis showed that BoPAG19 protein encoded 380 amino acids,the highest content serine (Ser) was 9.2%,and the lowest content tryptophan (Trp) was 1.6%.The molecular formula of BoPAG19 protein was C1937H3028N524O532S15,with theoretical molecular weight of 41.8 ku,isoelectric point of 9.62,instability index of 40.75,instability in water,fat coefficient of 91.53.The extinction coefficient was 52 370 mol-1·cm-1,and it was water-soluble.There was 1 signal peptide rigion and without transmembrane region.There were 6 potential glycosylation sites and 11 B cell epitopes.The secondary structure of BoPAG19 protein was composed of alpha helix (18.95%),beta turn (6.32%),random coil (42.37%) and extended chain (32.37%),respectively.The tertiary structure prediction result was consistent with the secondary structure.Proteins interacting results showed that BoPAG19 had interactions with APLP2 and APP,which might be involved in neuroregulation during pregnancy.【Conclusion】 In this study,BoPAG19 gene was successfully expressed and purified,and the bioinformatics characteristics of BoPAG19 were analyzed,which laid a foundation for the study of structure,function and diagnosis of BoPAG19 protein.
Cloning,Bioinformatics and Expression Analysis of Bovine CCR7 Gene
SHEN Xiang, CHEN Linghui, YAO Jingjie, WANG Sanhu, BAI Yueyu, ZHANG Xiaojian
2022, 49(4):  1244-1252.  doi:10.16431/j.cnki.1671-7236.2022.04.005
Abstract ( 223 )   PDF (2016KB) ( 71 )  
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【Objective】 This study was aimed to explore the biological characteristics of the protein encoded by CC chemokine receptor 7(CCR7) gene and the expression of mRNA in breast tissues of healthy and inflammatory dairy cows.【Method】 The CDS region of CCR7 gene in Chinese Holstein dairy cows was amplified by PCR method and cloned,the similarity alignment and phylogenetic tree construction were carried out after sequencing.The composition,physicochemical properties and structure of CCR7 protein were analyzed by bioinformatics softwares.The expression difference of CCR7 gene in breast tissues between healthy and inflammatory dairy cows were detected by Real-time quantitative PCR.【Result】 The full-length of CCR7 gene CDS region in Chinese Holstein dairy cows was 1 325 bp,which had high similarity (95.2%) and close genetic relationship with sheep;And had low similarity (56.4%) and far genetic relationship with chicken.CCR7 gene encoded 379 amino acids,among which the content of leucine was the most,the content of valine was the second,and the content of histidine and tryptophan were the lowest.The molecular weight of CCR7 protein was 14.56 ku,the theoretical isoelectric point was 8.89,and the average hydrophilic coefficient was 0.640,it was a transmembrane hydrophobic protein.The cleavage site of signal peptide might exist between amino acids 24 and 25.The structure of alpha helix was the most in the secondary structure,as high as 51.72%,and the tertiary structure was mainly alpha helix and random coil.The expression of CCR7 gene in breast tissue of inflammatory dairy cows was extremely significantly higher than that in healthy dairy cows (P<0.01),which might play an important role in the immune regulation of dairy cows mastitis.【Conclusion】 The CDS region of bovine CCR7 gene was successfully cloned,and the corresponding bioinformatics analysis and tissue quantitative expression were carried out,which provided materials for further study of the specific functions of CCR7 gene,and was of great significance to explore the regulatory function of CCR7 gene in dairy cow mastitis.
Establishment of BHK-21 Cell Strain for Stably Expressing T7 RNA Polymerase
CHEN Yujing, ZHANG Yiyi, HUANG Zhengyang, SHI Jianzhou, LIU Yangkun, QIU Reng, YAO Lunguang, LI Na
2022, 49(4):  1253-1261.  doi:10.16431/j.cnki.1671-7236.2022.04.006
Abstract ( 519 )   PDF (4918KB) ( 121 )  
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【Objective】 BHK-21 cell line stably expressing T7 RNAP was constructed by establishing a lentivirus vector system overexpressing T7 RNA polymerase (T7 RNAP).【Method】 In this study,T7 RNAP gene was amplified using the primers designed according to Escherichia coli BL21 (DE3) genome sequence published in GenBank (accession No.:NZ_CP081489.1),and then cloned into lentiviral vector to obtain the recombinant plasmid pCDH-CMV-T7 RNAP.After screening positive clones and sequencing identification,the packaging system and auxiliary packaging plasmid containing recombinant plasmid pCDH-CMV-T7 RNAP were co-transfected into HEK-293T cells by liposome,and the lentivirus was packaged and purified to obtain a recombinant lentivirus with high virus titer.The collected virus solution was infected with BHK-21 cells with multiplicity of infection (MOI)=1,5,10 and 20 respectively.The fluorescence was observed after 72 h to screen the best MOI.The recombinant lentivirus was infected with BHK-21 cells under the optimal MOI conditions.The positive rate of infected cells was observed by the expression of green fluorescent protein copGFP.4 μg/mL puromycin was used as the resistance screening material for multiple rounds of screening,and finally BHK-21-T7 RNAP cell line was obtained.Further,three pairs of detection primers were designed for RT-PCR detection of the selected cell lines,and the ability of T7 RNAP to drive the downstream gene of T7 promoter was reflected by detecting the activity difference of sea kidney luciferase (hRluc) reporter gene between test and blank control groups.【Result】 T7 RNAP gene in the genome of Escherichia coli BL21 (DE3) cells was successfully amplified,the recombinant lentivirus vector was successfully constructed,and the titer of the recombinant lentivirus was 1.0×108 TU/mL.BHK-21-T7 RNAP cell line was successfully screened.The specific DNA sequence of T7 RNAP could be detected by RT-PCR.After transfection of plasmid containing T7 promoter driven hRluc (pT7-hRluc) into this cell line,it was found that the activity of hRluc in BHK-21-T7 RNAP cell line was extremely significantly higher than that blank control BHK-21 cells (P<0.01).【Conclusion】 In this study,BHK-21 cell lines stably expressing T7 RNAP were obtained,and T7 RNAP in the selected BHK-21-T7 RNAP cell lines could drive the transcription of the downstream gene hRluc of pT7 promoter.The results laid a cellular foundation for the establishment of RNA virus reverse genetics platform.
Bioinformatics and Gene Expression Analysis of miR-10b-5p in Different Tissues of Chickens
HUANG Zhengyang, ZHAO Zhenhua, HUANG Huayun, LI Chunmiao, WANG Qianbao, MU Chunyu, LI Shoufeng
2022, 49(4):  1262-1270.  doi:10.16431/j.cnki.1671-7236.2022.04.007
Abstract ( 192 )   PDF (3439KB) ( 90 )  
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【Objective】 The target gene prediction,bioinformatics analysis and tissue expression of miR-10b-5p in Suqin No.3 chickens at different growth stages were carried out to explore its role in chicken muscle growth and development.【Method】 The miR-10b-5p sequence of vertebrates were obtained through scientific research and miRBase database,the position of miR-10b-5p was determined using the Ensembl database,the phylogenetic tree was constructed based on the miRNA precursor sequence.The target genes of miR-10b-5p were predicted by using miRDB,microT and TargetScan websites,then GO function and KEGG pathway analysis were performed to reveal the function of miR-10b-5p gene.The expression changes of miR-10b-5p were detected by Real-time quantitative PCR in brain,cerebellum,pituitary gland,chest muscle,leg muscle,ovary,heart,liver,spleen,lung and kidney of chickens at different growth stages.【Result】 A total of 59 miR-10b-5p sequences from 59 species of animals were retrieved by miRBase.Each animal had only one miR-10b-5p form.Gene mapping analysis revealed that chicken miR-10b-5p was located in HOX gene family cluster on chromosome 2.Alignment analysis of the mature sequence of miR-10b-5p in common species showed that the mature sequence of miR-10b-5p had high homology and was relatively conserved among species.The phylogenetic tree analysis showed that miR-10b in primates,rodents,birds,and fishes in mammals were clustered first,and then clustered together into one group,which indicated that miR-10b was conservative in the process of evolution.Target genes prediction revealed that miR-10b-5p had 300 target genes.GO function analysis showed that target genes were mainly enriched mainly to chromatin assembly and post-transcriptional regulation of gene expression,histone methyltransferase complexes,transcription factor complexes and other functions.KEGG pathway analysis showed that target genes were mainly enriched in Wnt and p53 signaling pathways.Tissue expression analysis showed that miR-10b-5p was detective in all tissues tested, the expression of miR-10b-5p in brain, cerebellum, pituitary, chest and leg muscles at 3-day-old were significantly higher than those in heart, hypothalamus, kidney and lung (P<0.05). The expression of miR-10b-5p in pituitary, chest muscle, leg muscle and brain were significantly higher than that in other tissues at 90-day-old (P<0.05).Compared with 3-day-old chicks,the expression of miR-10b-5p in pituitary,chest and leg muscles of chickens at 90-day-old was significantly increased (P<0.05).【Conclusion】 Chicken miR-10b-5p was a miRNA that was widely expressed in tissues,and miR-10b was conserved in the evolutionary process,it might regulate muscle growth and cell proliferation and differentiation through Wnt and p53 signaling pathways.
Physiological and Biochemical
Effect of miR-495-3p on Proliferation and Differentiation of Goat Skeletal Muscle Cells
YANG Lili, ZHANG Qiongwen, ZHANG Yu, LIU Xiaoxiao, CHEN Ting, JIANG Mingsheng
2022, 49(4):  1271-1279.  doi:10.16431/j.cnki.1671-7236.2022.04.008
Abstract ( 193 )   PDF (1406KB) ( 48 )  
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【Objective】 The experiment was conducted to study the effects of miR-495-3p on the proliferation and differentiation of goat skeletal muscle cells and its expression in different tissues,in order to provide a theoretical basis for exploring the regulatory mechanism of miRNA in muscle development.【Method】 Target genes of miR-495-3p were predicted by bioinformatics methods.The expression levels of miR-495-3p and its target genes in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and leg muscle were detected by Real-time quantitative PCR.The overexpression (miR-495-3p mimic,miR-495-3p mimic NC) and inhibitor (miR-495-3p inhibitor,miR-495-3p inhibitor NC) of miR-495-3p were constructed,and when the confluence of goat skeletal muscle cells reached 60%-70%,they were transfected and induced to differentiate with the medium containing 2% horse serum,CCK-8 was used to detect cell proliferation activity,and Real-time quantitative PCR was used to detect the overexpression and interference efficiency,and the expression of proliferation and differentiation related genes paired box 7 (Pax7),Cyclin E,myogenin (MyoG) and myogenic factor 5 (Myf5).The wild-type and mutant vectors of miR-495-3p target gene were constructed and transfected into 293T cells,the double luciferase report was used to verify the binding of miR-495-3p and its target gene.【Result】 Bioinformatics prediction results showed that the target gene of miR-495-3p was alpha cardiocactin 1 (ACTC1),and there were extremely significant differences of miR-495-3p and ACTC1 in longissimus dorsi muscle at the age of 1 and 10 months goats (P<0.01).miR-495-3p and ACTC1 were expressed in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and leg muscle,and the expression was higher in longissimus dorsi muscle.The results of cell transfection showed that miR-495-3p mimic significantly promoted the expression of miR-495-3p (P<0.01),and miR-495-3p inhibitor significantly inhibited the expression of miR-495-3p (P<0.01),indicating that miR-495-3p mimic and miR-495-3p inhibitor could be used in subsequent trials.Real-time quantitative PCR results showed that overexpression of miR-495-3p promoted the expression of skeletal muscle cell differentiation related marker genes MyoG and Myf5,while inhibition of miR-495-3p decreased the expression of MyoG and Myf5 genes.Overexpression and interference of miR-495-3p had no significant effect on the expression of Pax7,Cyclin E genes and cell proliferation activity of skeletal muscle cells (P>0.05).The results of double luciferase report showed that miR-495-3p had a targeting relationship with ACTC1 gene.【Conclusion】 miR-495-3p had no significant effect on the proliferation of goat skeletal muscle cells,but could promote the differentiation of goat skeletal muscle cells by targeting ACTC1 gene.
Effect of Tea Polyphenols on Proliferation and Migration of Mouse Melanoma Cell B16F10 in vitro
LIU Fangjun, LAN Wutao, LI Pengfei, XU Dongmei, ZHANG Lei, ZHU Zhiwei
2022, 49(4):  1280-1286.  doi:10.16431/j.cnki.1671-7236.2022.04.009
Abstract ( 225 )   PDF (1773KB) ( 88 )  
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【Objective】 This study was aimed to study the effects of tea polyphenols on the proliferation and migration of mouse melanoma cell B16F10,in order to provide evidence for the use of tea polyphenols in tumor therapy.【Method】 B16F10 cells were treated with different concentrations (0.05,0.10 and 0.15 g/L) of tea polyphenols,no treatment (0 g/L) was used as the control group.CCK-8 was used to determine the cell growth of different treatment groups,and the growth inhibition rate was calculated.After treated with different concentrations of tea polyphenols for 24 h,the migration ability of the cells was evaluated by scratch test.The expression of microphthalmia-associated transcription factor(MITF) and tyrosinase(TYR) mRNA and protein in B16F10 cells were detected by Real-time quantitative PCR and Western blotting.【Result】 Compared with control group,0.05 g/L tea polyphenols had no significant inhibitory effect on the proliferation of B16F10 cells (P>0.05),0.10 and 0.15 g/L tea polyphenols had extremely significant inhibitory effects on B16F10 cells (P<0.01),and the higher the concentration,the stronger the inhibition.0.05,0.10 and 0.15 g/L tea polyphenols had extremely significant inhibitory effects on the migration of B16F10 cells (P<0.01).Real-time quantitative PCR and Western blotting results showed that as the concentration of tea polyphenols increased,the expressions of MITF and TYR mRNA and protein gradually decreased in a concentration-dependent manner.Compared with control group,0.05 g/L tea polyphenols could significantly reduce the expressions of MITF mRNA and protein (P<0.05),0.10 and 0.15 g/L tea polyphenols could extremely significantly decrease the expression of MITF mRNA and protein (P<0.01).0.10 and 0.15 g/L tea polyphenols could extremely significantly decrease the expressions of TYR mRNA and protein (P<0.01).【Conclusion】 0.10 and 0.15 g/L tea polyphenols had significant inhibitory effect on the proliferation and migration of B16F10 cells,and could reduce the expression of melanogenesis genes MITF and TYR,indicating that tea polyphenols could inhibit the progression of melanoma.
Research Progress in Function of Snail Family Genes
LI Qilong, CHEN Xiangnan, LONG Feng, CHEN Jiayue, YANG Sen, WANG Jing, ZAN Linsen, CHENG Gong
2022, 49(4):  1287-1294.  doi:10.16431/j.cnki.1671-7236.2022.04.010
Abstract ( 444 )   PDF (727KB) ( 75 )  
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Snail family genes encode transcription factors with zinc finger structure,which participate in many physiological levels by combining downstream genes,and have a wide range of biological functions in epithelial-mesenchymal transformation,embryonic development,immune regulation,cancer cell migration and so on.Snail family genes are involved in the process of fat production and fat metabolism.At the same time,they are also the downstream target genes of myogenesis determinant (MyoD),and may also be involved in the regulation of muscle development.Therefore,Snail family genes play an important role in adipogenesis,muscle development and lipid metabolism,and they are important candidate functional genes affecting meat production performance and meat quality of mammals,especially agricultural animals.The author introduces the structure and basic biological function of Snail family genes,briefly describes the regulation mode of Snail family involved in Wnt/β-catenin,Notch and other signal pathways,and summarizes the role and regulation mode of Snail family genes in mammalian adipogenesis and muscle development.However,the role of Snail family genes in mammalian adipogenesis and muscle development remains to be further studied.On the other hand,it is generally believed that the Snail family,which plays a role of transcriptional inhibition,has also been found to have transcriptional activation in recent years,but how to achieve this effect is still unknown.Therefore,the future research direction is the synergism of Snail family genes in the regulation of animal adipogenesis and muscle development,the dynamic regulation of adipogenesis and fat hydrolysis and their transcriptional activation.This review lays a foundation for analyzing the genetic regulation mechanism of Snail family genes in the formation of meat quality traits.
Study on the Relationship Between OTUD7A Gene and Goose Fatty Liver Formation
SUN Qingyun, FAN Xiang, XING Ya, ZHAO Minmeng, LIU Long, GENG Tuoyu, GONG Daoqing
2022, 49(4):  1295-1303.  doi:10.16431/j.cnki.1671-7236.2022.04.011
Abstract ( 163 )   PDF (1943KB) ( 42 )  
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【Objective】 The experiment was aimed to study the relationship between the ovaries tumor deubiquitinase 7A (OTUD7A) gene and the formation of goose fatty liver.【Method】 40 70-day-old male Landes geese with similar body weight were selected and randomly divided into two groups.The geese of control group were free to feed,and the geese of experimental group were overfeeding.The daily intake was 500 g for the 1st to 5th day of filling,800 g for the 6th to 12th day,and 1 200 g for the 13th to 19th day.After overfeeding for 7,14 and 19 days,six geese in each group were randomly selected and slaughtered for liver samples.The expression levels of OTUD7A gene in livers of different overfeeding stages were determined by Real-time quantitative PCR.The goose primary hepatocytes were isolated from Landes goose embryos after 23 days of incubation using type Ⅳ collagenase digestion method.Goose primary hepatocytes were treated with 0 (blank control group),125 and 250 mmol/L glucose,0 (blank control group),50,100 and 200 nmol/L insulin,0 (blank control group),0.125 and 0.250 mmol/L oleic acid,linoleic acid,and 0 (blank control group),0.25 and 0.50 mmol/L palmitic acid,respectively,and the effects of these factors that related to fatty liver formation on the expression level of OTUD7A gene were detected by Real-time quantitative PCR.In addition,the overexpression vector pcDNA3.1-OTUD7A was constructed,and the constructed overexpression recombinant plasmid was transfected into goose primary hepatocytes,and the differentially expressed genes were screened by transcriptomic sequencing (RNA-Seq),and the obtained differentially expressed genes were analyzed by GO function enrichment.【Result】 OTUD7A gene expression in fatty liver was significantly higher than that in control group at days 7 and 14 of overfeeding (P<0.05).Compared to blank control group,OTUD7A gene expression in goose primary hepatocytes was up-regulated by 250 mmol/L of glucose,and 0.125 or 0.250 mmol/L of palmitate (P<0.05).Transcriptomic sequencing results revealed that a total of 34 differentially expressed genes were screened after OTUD7A gene overexpression,of which 19 genes were up-regulated and 15 genes were down-regulated.The GO function enrichment analysis of RNA-Seq showed that the entries were annotated as defense response to other organism,response to external biotic stimulus,T cell differentiation,extracellular exosome and so on.The expressions of CLMP,PROCR,SH3BP1,ARHGAP28,ACE,OTUD7A,LOC106045877 and LOC106048002 genes were up-regulated,and the expressions of TNFSF8,DDX60,PLAC8,RSAD2,MX1,RSAD2 and GBP1 genes were down-regulated.【Conclusion】 The expression level of OTUD7A gene was increased during the formation of goose fatty liver,OTUD7A might participate in the development of goose fatty liver through regulating the expressions of TNFSF8,RSAD2,MX1,GBP1,CLMP and PROCR.
Nutrition and Feed
Effects of Different Feeding Levels on Growth Performance,Slaughter Performance and Organ Development of Multiparous Suffolk Rams
NIU Yujie, ZHENG Xinxia, NIU Junli, ZHANG Wenju, CHEN Ning, LIU Yanfeng
2022, 49(4):  1304-1313.  doi:10.16431/j.cnki.1671-7236.2022.04.012
Abstract ( 244 )   PDF (931KB) ( 73 )  
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【Objective】 The aim of this experiment was to study the effect of feeding levels of ration on growth performance,slaughter performance,viscera and other organ development of Multiparous Suffolk rams.【Method】 18 Multiparous Suffolk rams in healthy condition with similar body weight (20.00 kg±1.50 kg) were randomly divided into three groups of six rams each.Group Ⅰ was fed ad libitum,group Ⅱ was fed 60% of the feed intake in group Ⅰ,and group Ⅲ was fed 40% of the feed intake in group Ⅰ.When the average body weight of the test sheep in group Ⅰ reached 35 kg,all test sheep were slaughtered and the growth performance,slaughter performance,and the weight and development of internal organs were measured.The pre-feeding period was 7 d,and the normal test period was 50 d.【Result】 ① With the decrease of feeding level of the ration,the dry matter intake,final weight,net weight gain and average daily weight gain of the experimental sheep were significantly decreased (P<0.05).The feed-to-gain ratio of groups Ⅰ and Ⅱ were significantly lower than that of group Ⅲ (P<0.05).②The carcass weight,net carcass meat weight and fat weight of group Ⅰ were significantly higher than those of groups Ⅱ and Ⅲ,and the difference between the groups was significant (P<0.05).The net meat rate of group Ⅰ was significantly higher than those of the other two groups (P<0.05).The bone weight,meat-bone ratio and abdominal fat weight of group Ⅰ were significantly higher than those of group Ⅲ (P<0.05).The abdominal fat rate of group Ⅲ was significantly lower than those of the other two groups (P<0.05).③The rumen weight was the highest in group Ⅰ and the lowest in group Ⅲ,and the difference between the groups was significant (P<0.05).The reticulum weight and total intestinal weight were the highest in group Ⅰ,the second highest in group Ⅱ and the lowest in group Ⅲ,and the difference between the 3 groups was significant (P<0.05).④Heart,liver,lung and kidney weights were the highest in group Ⅰ,followed by group Ⅱ and the lowest in test group Ⅲ,with significant differences among the groups (P<0.05).⑤The weight of head and hoof was the highest in group Ⅰ,which was significantly higher than that in test group Ⅲ (P<0.05),and the difference with test group Ⅱ was not significant (P>0.05).The weights of skin and hair,blood were the highest in test group Ⅰ,followed by test group Ⅱ,and the lowest in test group Ⅲ,and the difference between the three groups was significant (P<0.05).【Conclusion】 Different feeding levels could affect the growth performance,slaughter performance and development of major physiological organs of Multiparous Suffolk rams to different degrees.
Effects of Oregano Essential Oil and Colostrum Complex on Growth Performance, Serum Indicators Related Antioxidant and Immune in Opportunity Piglets
ZHANG Rui, ZHOU Weiqiang, MA Yue, WU Jianping, LEI Zhaomin
2022, 49(4):  1314-1321.  doi:10.16431/j.cnki.1671-7236.2022.04.013
Abstract ( 200 )   PDF (865KB) ( 74 )  
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【Objective】 This study was aimed to conduct the effects of oregano essential oil and colostrum complex (EC) with different levels on growth performance,serum biochemical indicators,antioxidant and immune capacity in opportunity piglets (body weight were <1.20 kg),further provide technical basis for promoting healthy growth of opportunity piglets.【Method】 A total of 128 binary sows (Landrace×Large White) with similar body condition,parity (2/3) and litter sizes were divided into 4 groups randomly,32 sows per group,all sows were fed as the same diet formula.Opportunity piglets from each sow group were administrated 0(control,CON),2(EC2,oral administration once),4(EC4,for 2 consecutive days) and 6 mL(EC6,for 3 consecutive days) EC by gavage after birth,respectively.The trial period lasted for 21 days from birth to weaning.The birth weight and weaning weight of all piglets were recorded to calculate average daily gain (ADG).Serum of opportunity piglets was collected at the end of experiment to measure the antioxidant and immune indexes.【Result】 Compared to CON,feeding EC after birth significantly increased weaning weight and ADG (P<0.05),and significantly decreased diarrhea indexes of opportunity pigs (P<0.05).The mortality in EC4 and EC6 decreased significantly (P<0.05).And the diarrhea indexes and mortality in EC4 were significantly lower than EC2 (P<0.05).The activities of ALT and AST in EC6 were significantly lower than CON (P<0.05),while the concentrations of GLB,IgA,IgG and IgM,and the activities of SOD and GSH-Px were significantly higher than CON and EC2 (P<0.05).【Conclusion】 In summary,neonatal opportunity piglets administered EC could improve growth performance,and continuous feeding for 2 days could significantly decrease diarrhea and death;While continuous feeding for 3 days could improve immune and antioxidant property.
Effects of Compound Chinese Medicine Fermentation Powder on Growth Performance,Nutrient Apparent Digestibility,Antioxidant Function and Immune Function of Weaned Piglets
WANG Chaopu, MA Xianyong, TIAN Zhimei, YU Miao, LIU Zhichang, DENG Dun, RONG Ting, TI Junling, SONG Min, ZHANG Huihua
2022, 49(4):  1322-1331.  doi:10.16431/j.cnki.1671-7236.2022.04.014
Abstract ( 231 )   PDF (1061KB) ( 73 )  
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【Objective】 This experiment was conducted to investigate the effects of compound Chinese medicine fermentation powder on growth performance,nutrient apparent digestibility,antioxidant function and immune function in weaned piglets.【Method】 48 healthy crossbred and castrated weaned piglets (Duroc×Landrace×Yorkshire) of 28-day-old with similar initial body weight of (8.290±0.140) kg were randomly divided into 2 groups with 6 replicates per group and 4 pigs per replicate.Pigs in the control group were fed a basal diet,while the piglets in the experimental group was fed a basal diet containing 3% compound Chinese medicine fermentation powder.The compound Chinese medicine fermentation powder was fermented from Hawthorn,Tangerine peel,chicken’s gizzard-membrane,Chinese yam and Poria cocos,and its main active substances include organic acids,flavonoids,polysaccharides and triterpenoids.The trial lasted for 28 d,3 d before the end of the experiment,dietary and fecal samples were collected.At the end of the experiment,one weaned piglets with an average body weight were selected from each replicate for slaughter,plasma and liver were collected and growth performance,nutrient apparent digestibility,plasma and liver antioxidant indices and plasma immune indices were detected.【Result】 Compared with the control group,there were no significant differences in the average daily feed intake (ADFI) and the feed to gain ratio (F/G) of weaned piglets in the compound Chinese medicine fermentation powder group (P>0.05),but the average daily gain (ADG) was significantly increased (P<0.05),and the diarrhea rate was significantly decreased (P<0.05).Compared with the control group,the apparent digestibility of ether extract (EE) and ash (Ash) and calcium (Ca) in the compound Chinese medicine fermentation powder group were extremely significantly or significantly increased (P<0.01;P<0.05).Compared with the control group,the plasma catalase (CAT) and glutathione peroxidase (GSH-Px) activities and the total antioxidant capacity (T-AOC) of weaned piglets in compound Chinese medicine fermentation powder group were significantly or extremely significantly increased (P<0.05;P<0.01).Furthermore,the activity of total superoxide dismutase (T-SOD) in liver of piglets in the compound Chinese medicine fermentation powder group was significantly higher than that of control group (P<0.05).Compared with the control group,the content of immunoglobulin A (IgA) and immunoglobulin M (IgM) in plasma of piglets in the compound Chinese medicine fermentation powder group were significantly increased (P<0.05),while the content of tumor necrosis factor-α (TNF-α) was significantly decreased (P<0.05).【Conclusion】 Adding 3% compound Chinese medicine fermentation powder in the basal diet could improve the apparent digestibility of EE,Ash and Ca,improve the antioxidation capacity and enhance the immune function of the body,thereby reducing diarrhea rate and improving the growth performance of weaned piglets.
Study on the Differences of Blood Indexes and Slaughter Performance Between 6-month-old Boer Goats and Macheng Black Goats
YANG Juan, GUO Shuai, HAN Shichang, ZHANG Nian, ZHANG Feng, TAO Hu, XIONG Qi
2022, 49(4):  1332-1342.  doi:10.16431/j.cnki.1671-7236.2022.04.015
Abstract ( 311 )   PDF (2490KB) ( 40 )  
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【Objective】 The differences of blood physiological and biochemical indexes,carcass traits,muscle fiber diameter and intramuscular fat content were researched between Boer goats and Macheng Black goats at the age of 6 months,so as to provide basis and reference for the feeding management,introduction improvement and cross utilization of the two breeds of goats.【Method】 8 healthy Boer goats and Macheng Black goats aged 3 months±5 days were bred under uniform conditions for 3 months.After feeding,blood was collected to detect 14 blood physiological indexes including white blood cell count (WBC),red blood cell count (RBC),hemoglobin concentration (HGB),hematocrit (HCT),average red blood cell volume (MCV) and so on,14 blood biochemical indexes including creatinine (CRE),total bilirubin (TBIL),urea nitrogen (BUN),blood glucose (GLU),total protein (TP) and so on,and 12 carcass traits including body weight,body height,body oblique length,carcass weight,slaughter rate,eye muscle area and so on.After slaughtered,the longissimus dorsi muscle tissue was taken and sliced,the muscle fiber diameter and intramuscular fat content were detected by hematoxylin and eosin staining and modified oil red O staining.【Result】 Among the blood physiological indexes,the hematocrit and average erythrocyte volume of Boer goats were significantly lower than those of Macheng Black goats (P<0.05),and the average erythrocyte hemoglobin concentration was significantly higher than that of Macheng Black goats (P<0.05).In blood biochemical indexes,the contents of total protein,globulin,blood phosphorus and amylase activity of Boer goats were extremely significantly lower than those of Macheng Black goats (P<0.01),and the content of potassium ion was significantly higher than that of Macheng Black goats (P<0.01).In carcass traits,the body weight,average daily gain,longissimus dorsi muscle weight,biceps femoris muscle weight and carcass weight of Boer goats were significantly higher than those of Macheng Black goats (P<0.05),the eye muscle area was extremely significantly larger than that of Macheng Black goats (P<0.01),and there was no significant difference in other indexes (P>0.05).The muscle fiber diameter of Boer goats was extremely significantly larger than that of Macheng Black goats (P<0.01),the content of intramuscular fat was significantly higher than that of Macheng Black goats (P<0.05).【Conclusion】 There were great differences in blood indexes and slaughter performance of Boer goats and Macheng Black goats.The results could provide a theoretical basis for the germplasm characteristics of Boer goat and Macheng Black goat.
Effects of Compound Nutrients on Growth Performance,Nutrient Apparent Digestibility and Serum Parameters of Fattening Sheep
HU Yuchao, GUO Tao, YANG Yi, YANG Lei, CUI Zhiwei, MAO Jinju, GUO Hui, WANG Chunyuan, WANG Yuan, AN Xiaoping, QI Jingwei
2022, 49(4):  1343-1351.  doi:10.16431/j.cnki.1671-7236.2022.04.016
Abstract ( 243 )   PDF (941KB) ( 103 )  
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【Objective】 The objective of this study was to evaluate the effects of compound nutrients mainly composed of fermented wheat bran polysaccharides,cinnamaldehyde,eugenol and capsicum oleoresin on growth performance,nutrient apparent digestibility and serum biochemical parameters of Dorper×Thin-tailed Han crossbred fattening sheep,expecting to provide a certain theoretical basis for the application of compound nutrients in the production of mutton sheep.【Method】 A single-factor randomized block design was adopted in the experiment,thirty-six 2-3-month-old Dorper×Thin-tailed Han crossbred lambs with body weight of (29.69±3.11) kg were randomly divided into 3 groups with 6 replicates per group and 5 lambs per replicate.Lambs in the control group (CON) were fed with basal diets,lambs in the groups Ⅰ and Ⅱ were fed with basal diets supplemented with 2.5 and 5.0 g/kg compound nutrients,respectively.The pre-experimental period lasted for 14 days,and the experimental period lasted for 60 days.The sheep were weighed at the beginning and ending of the experiment to determine their growth performance,blood samples were collected to determine serum physiological and biochemical indexes,antioxidant and immune ability.Fecal samples and feed samples were collected continuously for 7 days on the 24th to 30th day and 54th to 60th day of the trial period,once in the morning and once in the evening,to determine the apparent digestibility of nutrients.【Result】 ① Compared with control group,the final weight and average daily gain of the fattening sheep in group Ⅰ were increased significantly (P<0.05),and the feed-to-gain ratio was decreased,but the difference was not significant (P>0.05).The dry matter intake of the fattening sheep in the experiment groups Ⅰ and Ⅱ was significantly increased (P<0.05).② Compared with control group,the apparent digestibility of dry matter and organic compound of groups Ⅰ and Ⅱ were significantly increased on the 24th to 30th day (P<0.05).③ Dietary compound nutrients had no significant effect on serum biochemical parameters of fattening sheep (P>0.05).Compared with control group,at the end of the trial,the content of serum superoxide dismutase (SOD) and immunoglobulin M(IgM) in groups Ⅰ and Ⅱ were significantly increased (P<0.05),the contents of serum glutathione (GSH) and immunoglobulin G(IgG) in group Ⅱ were significantly reduced (P<0.05),the content of serum malondialdehyde (MDA) in group Ⅰ was significantly reduced (P<0.05).【Conclusion】 Adding 2.5 g/kg of compound nutrients to the diet could increase dry matter intake and apparent digestibility,improve immunity and increase the body’s antioxidant level of fattening sheep.
Genetics and Breeding
Genetic Diversity Analysis of Shaanxi Moschus berezovskii Based on mtDNA Cytb Gene and D-loop Region Sequence
LIU Xiaowei, LI Ruixiang, WANG Xinglong, LI Xiao
2022, 49(4):  1352-1363.  doi:10.16431/j.cnki.1671-7236.2022.04.017
Abstract ( 183 )   PDF (1998KB) ( 39 )  
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【Objective】 This study was aimed to explore the genetic diversity of Shaanxi Moschus berezovskii population,and understand the genetic information of Moschus berezovskii.【Method】 The hair of Moschus berezovskii was collected to extract DNA,the mitochondrial DNA(mtDNA) cytochrome b(Cytb) gene and D-loop sequences of 43 Moschus berezovskii individuals were determined,and the base composition was counted.All sequences were integrated and compared using ClustalX 2.0 software to obtain nucleotide polymorphic sites (SNPs) in the population.The nucleotide diversity (Pi),number of haplotype (H),haplotype diversity (Hd) and average number of nucleotide differences (K) were calculated by DNASP 5.10 software.The genetic distance among different haplotypes of Cytb gene and D-loop sequences was calculated by Mega 7.0 software,and Neighbor-Joining (NJ) phylogenetic tree was constructed.【Result】 The AT content of Cytb gene and D-loop region were higher than GC content,indicating there was bias in base composition.There were 241 and 383 SNPs of Cytb gene and D-loop region,respectively.The nucleotide diversity of Cytb gene and D-loop region were 0.28343 and 0.07707,and the haplotype diversity was 0.983 and 0.975,respectively,indicating that the population genetic diversity was rich.The genetic distances of 35 haplotypes of Cytb gene ranged from 0.002 to 0.831,and 29 haplotypes of D-loop region ranged from 0.006 to 1.342.The phylogenetic tree showed that there were two mitochondrial lineages,indicating that there were two mitochondrial maternal origins.The evolutionary analysis of D-loop region also supported this conclusion.【Conclusion】 The nucleotide diversity and haplotype diversity of Moschus berezovskii population were high,and the genetic diversity was rich.At the same time further supported the view of Moschus berezovskii and Moshus moschiferus belonged to a branch of the view.
Polymorphism of MyoG Gene Intron Ⅱ and Its Association with Growth Traits in Sheep
LI Jingyun, WANG Xinle, ZHANG Yunfei, BAI Junyan, LI Gan, LEI Ying, DONG Zhihao, CHEN Yu, FAN Hongdeng, WANG Longwei, CHEN Mengke, WANG Xingke, SIMA Leiyang, ZHANG Shiwen
2022, 49(4):  1364-1373.  doi:10.16431/j.cnki.1671-7236.2022.04.018
Abstract ( 167 )   PDF (2817KB) ( 39 )  
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【Objective】 This study was aimed to analyze the polymorphism of myogenin (MyoG) gene intron Ⅱ and its effect on growth traits in sheep,the molecular markers with significant effects on the growth and development of sheep were screened to provide reference for molecular breeding in sheep.【Method】 Five species of Large-tailed Han sheep,Small-tailed Han sheep,Yuxi Fat-tailed sheep,Dorper sheep and Hu sheep were selected as the research object. The intron Ⅱ of MyoG gene (Eco72 Ⅰ) was genotyped by PCR-RFLP technology.The population genetic polymorphism of MyoG gene intron Ⅱ was calculated by PopGene32 software.The association analysis between the different genotypes of MyoG gene intron Ⅱ and growth traits of sheep was performed by SPSS 17.0 software.【Result】 There were three genotypes of AA (368/540 bp),AB (908/368/540 bp) and BB (908 bp) were found of MyoG gene intron Ⅱ in Small-tailed Han sheep,Dorper sheep and Hu sheep populations,and there were two genotypes of AB (908/368/540 bp) and BB (908 bp) were found in Large-tailed Han sheep and Yuxi Fat-tailed sheep populations.The genotype frequency of AB in Large-tailed Han sheep,Small-tailed Han sheep,Yuxi Fat-tailed sheep,Hu sheep and Dorper sheep were 0.845,0.633,0.917,0.706 and 0.811,the genotype frequency of BB were 0.155,0.033,0.083,0.176 and 0.054,and the genotype frequency of AA genotype were 0,0.333,0,0.118 and 0.135,respectively.The lowest heterozygosity was 0.455 in Small-tailed Han sheep,and the highest was 0.497 in Dorper sheep,indicating that Dorper sheep had a higher genetic diversity than other population.The polymorphic information content (PIC) of five sheep population was moderate polymorphism (0.25<PIC<0.5).The association analysis results showed that the chest circumference,chest width,head length and neck length of BB genotype of MyoG gene intron Ⅱ in Small-tailed Han sheep were significantly lower than those of AA and AB genotypes (P<0.05).【Conclusion】 The Eco72 Ⅰ site of MyoG gene intron Ⅱ could be used as a molecular marker affecting the growth trait in sheep,and the results provided reference for molecular marker assisted selection of growth trait in sheep in the future.
Research Advances on CRISPR/Cas9 Technology in Livestock and Poultry Breeding
XU Jing, YANG Guang, JIANG Meiqi, DING Xiangbin, GUO Yiwen, HU Debao, LI Xin, GUO Hong, ZHANG Linlin
2022, 49(4):  1374-1383.  doi:10.16431/j.cnki.1671-7236.2022.04.019
Abstract ( 277 )   PDF (1100KB) ( 150 )  
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When using traditional methods to modify the genome at a specific locus of livestock and poultry,it can only be realized by homologous recombination in somatic cells and nuclear transfer.The difficulty and low efficiency of traditional homologous recombination methods hinder the wide application of gene modification in livestock and poultry genetics and breeding.In recent years,the discovery of site-specific endonucleases provides a different and more direct way for targeted gene modification,because these enzymes can directly carry out one-step gene editing on DNA sequence.Clustered regularly interspersed short palindromic repeat/CRISPR associated protein 9 (CRISPR/Cas9) is an RNA oriented DNA endonuclease,which can accurately locate specific target sites and efficiently complete RNA oriented DNA recognition and editing.As an accurate and powerful third-generation genome editing tool,CRISPR/Cas9 technology has been successfully applied to pigs,cattle,goats,sheep and chickens.These CRISPR/Cas9 gene edited livestock can be used as biological models to study human or livestock and poultry physiology and pathology model,bioreactors to produce functional proteins or donors for organ transplantation.Especially in livestock and poultry production,CRISPR/Cas9 genome editing can be used to improve the genetic characteristics of production,improve the quality of livestock and poultry products and the resistance of livestock and poultry to diseases.The author summarized the principle of CRISPR/Cas9 technology with specific site genome modification in livestock and poultry and the latest progress of its genome editing in livestock and poultry breeding,in order to provide reference for promoting the research of CRISPR/Cas9 technology in livestock and poultry breeding.
Correlation Analysis of VNN1 Gene Polymorphism and Lambing Number in F3 Generation Boza Goats
RUAN Yong, CHEN Xiang, DAI Lingang, HUANG Jiajin, AN Dongwei, ZOU Qishun
2022, 49(4):  1384-1392.  doi:10.16431/j.cnki.1671-7236.2022.04.020
Abstract ( 187 )   PDF (3827KB) ( 25 )  
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【Objective】 This experiment was conducted to investigate the relationship between panthenoyl mercaptoethamine-1 (VNN1) gene polymorphism and lambing number of F3 generation Boza goats,so as to provide molecular guidance for breeding of F3 generation Boza goats.【Method】 107 F3 generation Boza goats were selected for this experiment,blood samples were collected to extract DNA.According to the exon sequence of VNN1 gene(accession No.:NC_030816.1),7 pairs of primers were designed for PCR amplification and sequencing.DNAStar software was used to analyze the sequencing results,and PCR amplification and sequencing were performed on the entire group of possible single nucleotide polymorphism (SNP) sites.Hardy-weinberg equilibrium and genetic diversity analysis were carried out,and the correlation analysis between the different genotypes of VNN1 gene and the lamb number in F3 generation Boza goats was conducted by SPSS 16.0 statistical software.【Result】 There were 7 SNPs in exons 5 and 7 of VNN1 gene (g.10956 A→G,g.11010 C→T,g.11103 C→T,g.11284 C→A,g.11313 T→C,g.18094 C→T and g.18158 G→C),and 3 different genotypes existed in each site.χ2 test showed that g.10956 A→G,g.11010 C→T,g.11103 C→T,g.11313 T→C and g.18094 C→T were in Hardy-Weinberg equilibrium.g.11284 C→A and g.18158 G→C were hardy-Weinberg disequilibrium.The polymorphic information content was moderate polymorphis (0.25<PIC<0.5).Correlation analysis showed that g.11010 C→T and g.11313 T→C were significantly correlation with the lamb number in F3 generation Boza goats,CC genotype of g.11010 C→T was lower than CT and TT genotypes;TT genotype of g.11313 T→C was significantly lower than TC and CC genotypes (P<0.05).There was no significant correlation between g.10956 A→G,g.11103 C→T and g.18158 G→C and the lamb number in F3 generation Boza goats (P>0.05).【Conclusion】 There were 7 SNPs in exons 5 and 7 of VNN1 gene,g.11010 C→T and g.11313 T→C of VNN1 gene were significantly correlated with the number of lambs in F3 generation Boza goats,and VNN1 gene could be used as a candidate gene.
Study on Genetic Evolution Mechanism of Daweishan Mini Chicken Based on RAD-Seq Technology
ZHANG Huiyong, LI Guohui, XUE Qian, ZHOU Chenghao, YIN Jianmei, SU Yijun, XIA Shuli, HAN Wei
2022, 49(4):  1393-1401.  doi:10.16431/j.cnki.1671-7236.2022.04.021
Abstract ( 169 )   PDF (1452KB) ( 30 )  
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【Objective】 This study was aimed to explore the genetic evolution of Daweishan Mini chicken at molecular level.【Method】 Daweishan Mini chicken and other 18 indigenous chicken breeds,as well as two introduced breeds were used as research objects.10 roosters and 20 hens were selected from each breed.The whole genome DNA was extracted from blood samples,and simplified genome sequencing(RAD-Seq) was carried out to identify genome SNP of 21 breeds.Analysis of genetic structure and diversity of Daweishan Mini chicken,Calculate the genetic statistics,analyze genetic diversity and structure,screen selected genes and perform functional enrichment analysis.【Result】 The results showed that 331 892 SNPs markers were identified in Daweishan Mini chicken.The population genetic analysis indicated that Daweishan Mini chicken possessed moderate genetic diversity,with the average heterozygosity (Ho,0.219) and nucleotide diversity (Pi,0.245),inbreeding coefficient(Fis,0.107).The results of cluster analysis showed that Daweishan Mini chicken had closer genetical affinity with Piao chicken,Chahua chicken and Tibetan chicken.Daweishan Mini chicken had the lowest Fst(0.0929) with Piao chicken and the highest Fst(0.2179) with Henan Game chicken,which indicated that Daweishan Mini chicken had the closest relationship with Piao chicken and farthest genetic relationship with Game chicken.A total of 200 selected genes were screened out,GO analysis showed that these selected genes were mainly enriched in the biological processes of locomotion,stimulus response and signal transduction.KEGG analysis showed that these genes were mainly enriched in signaling pathways including metabolism,regulation of actin cytoskeleton and MAPK.【Conclusion】 The genetic diversity of Daweishan Mini chicken was moderately polymorphic,and it was closely related to Piao chicken.200 selected genes were screened,which would play roles in metabolism,signal transduction and skull development.
Cloning of CNP Gene and Its Tissue Expression Characteristics at Puberty in Dolang Sheep
ZHANG Zhishuai, SUI Zhiyuan, ZHANG Jihu, LI Qingjin, XING Feng
2022, 49(4):  1402-1412.  doi:10.16431/j.cnki.1671-7236.2022.04.022
Abstract ( 184 )   PDF (4392KB) ( 23 )  
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【Objective】 The C-type natriuretic peptide (CNP) gene of Dolang sheep was cloned and analyzed,and its expression pattern in hypothalamus,pituitary gland,oviduct,ovary and uterus before and after the onset of puberty in Dolang sheep was explored,in order to provide a reference for studying the mechanism of CNP gene in the onset of puberty.【Method】 Primers were designed with reference to the sequence of sheep CNP gene in GeneBank (accession No.:XM_027974523.1),and RT-PCR technique was used to amplify CNP gene of Dolang sheep before,during and after the puberty,and cloning and sequencing were performed.DNAMAN software was used to compare similarity with other species and Mega 5.0 was used to construct phylogenetic tree.The nucleotide sequence of CNP gene and the physicochemical properties such as hydrophobicity,signal peptide,phosphorylation site,transmembrane domain,secondary and tertiary structures of the encoded proteins were analyzed using bioinformatics softwares.The expression of CNP gene in hypothalamus,pituitary,oviduct,ovary and uterus of Dolang sheep was examined using Real-time quantitative PCR technique.【Result】 The sequence size of the CNP gene of Dolang sheep was 2 227 bp,including 5'-UTR 50 bp,3'-UTR 914 bp and CDS region 1 263 bp.Similarity comparison and phylogenetic tree results showed that Dolang sheep had the closest affinity to goat and the most distant affinity to chicken.The CNP gene of Dolang sheep encoded a total of 420 amino acids and its encoding protein was a hydrophilic protein with no transmembrane structural domain or signal peptide.The secondary structure of the CNP protein in Dolang sheep was mainly α-helix and random coil.The tertiary structure prediction showed that the CNP protein had no ligand structure.Real-time quantitative PCR results showed that the CNP gene was significantly more expressed in the hypothalamus than in other tissues at pre-puberty,puberty and post-puberty (P<0.05),CNP gene expression in the hypothalamus was significantly lower in the puberty than in the pre-puberty phase (P<0.05).In uterus,the expression level of CNP gene was significantly higher in pre-puberty than puberty and post-puberty (P<0.05).【Conclusion】 The CNP gene was successfully cloned in Dolang sheep,which was mainly expressed in hypothalamus and uterus.The expression of CNP gene in hypothalamus tissue decreased and then increased during the developmental process pre-puberty,puberty and post-puberty suggesting that CNP gene might be involved in the regulation of the initiation process during pubertal phase.
Screening of Specific SNP Sites in Tahe Red Deer Based on Resequencing Technology
DENG Yihua, WANG Tianjiao, WANG Hongliang, DONG Yimeng, LIU Xin, XING Xiumei
2022, 49(4):  1413-1421.  doi:10.16431/j.cnki.1671-7236.2022.04.023
Abstract ( 244 )   PDF (1502KB) ( 145 )  
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:【Objective】 To screen high quality single nucleotide polymorphic site (SNP) of Tahe red deer,construct specific molecular genetic markers, provide reference for pure breed identification of Tahe red deer. 【Method】 The whole genome of 32 blood DNA samples of Tahe red deer collected by the National Special Economic Animal Resource Sharing Platform in 1999 was re-sequenced. Compared with the chromosome-level reference genome of sika deer, the alignment rate, coverage and sequencing depth were counted. SNPEff was used to count the distribution of SNP on each chromosome, SNPhylo was used to construct a molecular evolutionary tree based on the sites, VCFTOOLS was used to calculate the genetic differentiation index(Fst), and the Fst value was sorted in descending order and the threshold was settet as Fst≥0.25 to eliminate the unqualified loci. R language was used for principal component analysis, the top 1 500 SNPs of 33 chromosomes was counted, the eigenvalues of the top 100 SNPs was extracted, and the PCA cluster diagrams were drew respectively. 【Result】 The results of whole-genome resequencing showed that the effective data volume of the blood genomic DNA of 32 Tahe red deer that passed the quality inspection was 868 791 354 600 bp, and the sequencing quality met the requirements of subsequent data analysis. The average comparison rate of the 32 samples sequencing data was 98.06%, the average coverage was 97.66%, and the average depth was 6.787. After filtering, 20 139 122 high-quality SNPs were obtained, of which SNPs were the most distributed on chromosome 4, and more than 90% of the mutations were located in the intergenic and exon regions. The number of candidate specific SNP sites after establishment was 12 050 781. 544 717 SNPs were selected by VCFTOOLS. The top 1 500 SNPs of each chromosome were selected for PCA, the PCA results showed that when the number of SNP was decreased from 49 500 to 100, the discrimination efficiency was not decreased. Finally, 100 SNPs with strong specificity and high stability were screened out.【Conclusion】 100 Tahe red deer-specific SNPs were obtained through whole-genome resequencing technology and bioinformatics analysis, which provided a theoretical basis for the identification of purebred Tahe red deer and the screening of core germplasm.
Research Progress on Effects of Polyunsaturated Fatty Acids on Animal Semen Quality
YUAN Chongshan, WANG Jun, LYU Wenfa
2022, 49(4):  1422-1429.  doi:10.16431/j.cnki.1671-7236.2022.04.024
Abstract ( 230 )   PDF (992KB) ( 74 )  
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Polyunsaturated fatty acids (PUFAs) are mainly divided into ω-3 PUFA and ω-6 PUFA,which are the main components of sperm plasma membrane lipid and have the effect of improving antioxidant capacity,sperm plasma membrane fluidity,sperm structural integrity and testosterone synthesis.Arachidonic acid (AA) in ω-6 PUFA could promote the secretion of testosterone,but excessive AA in seminal plasma may induce oxidative stress;Linoleic acid (LA) can also increase the level of testosterone in the body,conjugated linoleic acid (CLA) can reduce the oxidative stress of sperm;A small amount of docosapentaenoic acid (DPA) in animals can not only participate in the transport process of sperm,but also improve the antioxidant capacity of sperm.Alpha-linolenic acid (ALA) in ω-3 PUFA has antioxidant effects and can stimulate the synthesis of testosterone,docosahexaenoic acid (DHA) is similar to ALA,can improve the antioxidant capacity of sperm,at the same time can promote testosterone synthesis;Eicosapentaenoic acid (EPA) can regulate sperm structure and testosterone synthesis.An appropriate ω-6/ω-3 PUFA ratio has important regulatory effects on sperm motility,plasma membrane structure,lipid composition and acrosomal response.The effect of PUFA on semen quality may vary depending on the method of addition,type,and proportion of ω-6/ω-3 PUFA.In recent years,great progress has been made in the research of PUFA on animal semen quality,but its mechanism and related approaches to improve semen quality still need to be further explored.In this paper,the effects of PUFA on animal semen quality were summarized,and the effects of different types of ω-3 PUFA,ω-6 PUFA and ω-6/ω-3 PUFA ratio on sperm were summarized,in order to provide theoretical reference for further understanding the application of PUFA in animal semen production.
Study on Porcine ICSI Embryo Sex Identification Based on Raman Spectroscopy and SVM
SUN Junli, SUN Ruyu, LIAO Haihong, LU Jianqin, YAN Jing, LU Shengsheng, ZHANG Bing
2022, 49(4):  1430-1437.  doi:10.16431/j.cnki.1671-7236.2022.04.025
Abstract ( 180 )   PDF (1850KB) ( 62 )  
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【Objective】 The purpose of this study was to establish a non-destructive,non-invasive and rapid method for determining the sex of embryos.【Method】 207 porcine intracytoplasmic sperm injection (ICSI) embryos and their culture medium were used as the research objects.After the sex of a single embryo was identified by nest PCR amplification,it was randomly divided into training set and test set.At the same time,the Raman spectrum of a single ICSI embryo culture medium was obtained by Raman spectroscopy.After the original spectrum was processed,the support vector machine (SVM) algorithm was used to construct the classification model.Firstly,the training samples were used for training and modeling,and then the gender of the test samples was predicted.Combined with the gender identification results of PCR,the classification accuracy was calculated.【Result】 The sex of 207 embryos was indentified by nest PCR, 71 male and 128 female was successfully identified,and 8 samples failed to be amplified.The characteristic peak intensity of porcine ICSI male embryo culture medium at 1 082 and 1 360 cm-1 Raman shifts was significantly higher than that of female embryo’s.The classification accuracy of the constructed embryo sex identification model was 81.5%,and the classification accuracy of female and male embryos were 81.3% and 81.8% respectively.【Conclusion】 The new and non-destructive method of embryo sex identification based on the Raman spectroscopy and SVM algorithm was constructed,and its classification accuracy was 81.5%.
Preventive Veterinary Medicine
Subcellular Localization and Screening of Host Interacting Proteins of Brucella Secreted Protein BPE005
QIU Runhui, GUAN Feihu, TAO Tingting, LI Jia, ZHAO Tianyi, DENG Xingmei, SHI Chao, SUN Zhihua, ZHANG Hui
2022, 49(4):  1438-1448.  doi:10.16431/j.cnki.1671-7236.2022.04.026
Abstract ( 184 )   PDF (8876KB) ( 52 )  
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【Objective】 This study was aimed to analyze the protein structure and cellular localization of Brucella secretory protein BPE005,and screen the target proteins that interact with it in host cells,so as to lay a foundation for the follow-up study of the biological function of BPE005.【Method】 The protein structure of BPE0005 was analyzed by bioinformatics software,the recombinant fluorescent localization vector of PDRRED2-C1-BPE005 was constructed and transfected into human renal epithelial cells 293T,the cellular localization of BPE005 was observed by scanning laser confocal microscope.The PGBKT7-BPE005 bait vector was constructed to verify whether the bait vector had cytotoxicity and self-activation.The cDNA library of mouse macrophage RAW264.7 mRNA was used as AD library,and the titer of the library was detected.The target proteins interacting with BPE005 in host cells were screened by yeast two-hybrid system.【Result】 Brucella secretory protein BPE005 was hydrophobic and contained a large number of random coil,alpha helix and extended chain.The PDRRED2-C1-BPE005 fluorescence localization vector and PGBKT7-BPE005 bait protein vector were successfully constructed.BPE005 was located in the host nucleus.The PGBKT7-BPE005 bait protein vector had no yeast cytotoxicity and self-activation.The titer of AD library was more than 2×107/mL.Four host cell proteins interacting with BPE0005,RNA polymerase Ⅱ polypeptide G,complement factor H,guanylatecyclase 2G and granule protein were screened.After rescreening,sequencing,BLAST comparison and one-on-one verification,a target protein (RNA polymerase Ⅱ peptide G) with strong interaction with BPE005 in host cells was selected.The analysis of the action network of the protein showed that RNA polymerase Ⅱ peptide G had a significant effect on the transcription and synthesis of mRNA in host cells.【Conclusion】 Brucella secretory protein BPE005 was located in the host nucleus,and could interact strongly with polypeptide G of RNA polymerase Ⅱ in host cells,which was of guiding significance for further elucidating the action mechanism of key effector molecules in the process of Brucella infection.
Infectivity of H9N2 Subtype Avian Influenza Virus SD18 Strain and Comparison of the Effects of Different Routes of Infection
HOU Xiaohong, WANG Meng, WU Bo, LONG Xianrong, ZHAO Xiaomin, GAO Jing, DAI Peiqiang, CHAI Tongjie
2022, 49(4):  1449-1460.  doi:10.16431/j.cnki.1671-7236.2022.04.027
Abstract ( 257 )   PDF (2126KB) ( 146 )  
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【Objective】 The study was aimed to elucidate the transmission and pathogenicity of H9N2 subtype Avian influenza virus (AIV) in mammals.【Method】 Samples were collected for virus isolation and identification in this study.The isolated virus was used to infect guinea pigs by nasal drops and lung delivery at a dose of 300 μL (107 EID50),with 15 guinea pigs in each group,and the infection effects of the two groups were compared,then the aerosol transmission ability of the isolated strain was verified by the transmission ability experiment in guinea pigs.The replication ability of the isolated strain on human bronchial epithelial cells was detected by indirect immunofluorescence assay.【Result】 A strain of H9N2 subtype AIV was isolated and was named SD18.After guinea pigs were infected with the virus by nasal drip and lung delivery,the guinea pigs infected by lung delivery showed greater virus excretion than the nasal drip (P<0.05).Through the detection the expression of pattern recognition receptors and antiviral proteins in lungs of guinea pigs,the expression of immunologic factors Toll-like receptor 3 (TLR3),TLR7,myxovirus-resistance (MX),2'-5'oligoadenylates synthesis (OAS) had extremely significantly increased in both modes of infection (P<0.01).Antibodies of H9N2 subtype AIV began to be developed on the 6th day and showed an upward trend.The transmission ability of SD18 strain in guinea pigs was verified.It was found that SD18 strain had the ability to infect guinea pigs through direct contact and droplet transmission,but the generation and transmission of aerosols had not been confirmed.When human bronchial epithelial BEAS-2B cells were infected with SD18 strain,the virus could be detected at 12 h after infection,its replicability within cells peaked at 24 h,and the expression of immune factors TLR3,TLR7,MX,OAS,interleukin 6 (IL-6),IL-8 and interferon β(IFN-β) were extremely significantly upregulated (P<0.01).【Conclusion】 The interspecies transmission of SD18 strain had considerably become stronger,it could directly infect guinea pigs without passage adaptation.SD18 strain had the ability to infect guinea pigs and produce antibodies through droplets,and SD18 strain could replicate on human bronchial epithelial BEAS-2B cells.
Effects of Trichomonas gallinae Infection in Breeding Pigeons on Growth Performance, Carcass Traits and Immune Function of Squab in Pigeon
BIAN Shixiong, LI Yunlei, NI Aixin, GE Pingzhuang, WANG Yuanmei, ZONG Yunhe, HAN Pengmin, ZHAO Jinmeng, YUAN Jingwei, SUN Yanyan, MA Hui, CHEN Jilan
2022, 49(4):  1461-1469.  doi:10.16431/j.cnki.1671-7236.2022.04.028
Abstract ( 299 )   PDF (15354KB) ( 38 )  
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【Objective】 The purpose of this experiment was to study the harm to squab caused by Trichomonas gallinae infection of breeding pigeons and provide reference for prevention and control of Trichomonas gallinae disease in pigeon farm.【Method】 Eighteen pairs of uninfected and eighteen pairs of infected pigeons were selected as control group and experimental group,and each pair of pigeons fed two squabs.The experiment lasted for 28 days.The death rate,growth performance,carcass performance,immune organ index and antibody levels of squabs were measured.【Result】 The number of Trichomonas gallinae in experimental group was significantly higher than that in control group (P<0.05),and it was increased with the age of squabs.The death rate of squab in experimental group was 26.92%,which was significantly higher than that in control group (P<0.05).The average daily gain of squabs in experimental groups from 1 to 7 days was significantly lower than that in control group (P<0.05),but there were no significant differences in outbound weight and carcass performance (P>0.05).Trichomoniasis could cause pathological damage to oral mucosa,thymus,liver and spleen,and the contents/activities of ALB,ALT and AST in blood were significantly increased (P<0.05),while the level of sCD8 was significantly decreased (P<0.05).【Conclusion】 Infection of Trichomonas gallinae in breeding pigeons could cause squab infection,cause obvious pathological damage to the tissues such as liver and spleen of squab,reduce immunity of squab,and increase mortality of squab.
Preparation of Quercetin-tilmicosin Mixture Nanoparticles and Its Antibacterial Activity Against Small Colony Variant Strains of Staphylococcus aureus
LIU Jinhuan, ZHANG Shanling, SONG Wei, LUO Wanhe, CHEN Wei
2022, 49(4):  1470-1478.  doi:10.16431/j.cnki.1671-7236.2022.04.029
Abstract ( 189 )   PDF (3205KB) ( 31 )  
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【Objective】 Quercetin-tilmicosin mixture nanoparticles was prepared for the treatment of Staphylococcus aureus small colony variants (SASCVs) infection,and its antibacterial activity against SASCVs strain was evaluated.【Method】 The dosage of quercetin (0.05,0.10,0.15 and 0.20 g) and tilmicosin API (0.2,0.3,0.4 and 0.5 g),rotating speed (500,1 000 and 1 500 r/min),reaction time (0.5,1.0 and 2.0 h),reaction temperature (25,50 and 75 ℃) and solvent concentration (5.0%,7.5% and 10.0%) were determined to screen the optimal formula and preparation conditions of quercetin-tilmicosin mixture nanoparticles.The prepared quercetin-tilmicosin mixture nanoparticles were characterized by micro-morphology,sedimentation volume ratio,re-dispersibility,particle size and Zeta potential.The antibacterial activities of quercetin-tilmicosin mixture nanoparticles and commercial tilmicosin solution against SASCVs strains were compared by agar diffusion method,micro broth dilution method and in vitro sterilization curve,and the advantages of quercetin-tilmicosin mixture nanoparticles were evaluated.【Result】 Quercetin-tilmicosin mixture nanoparticles were the best when tilmicosin was 0.4 g,quercetin was 0.1 g,solvent was 20 mL of 7.5% polyethylene glycol solution,rotating speed was 1 000 r/min,reaction time was 1 h and reaction temperature was 50 ℃,respectively.Its appearance properties of formulation showed no precipitate and floccule,and the color was clear.Its micro-morphology showed that the mixture nanoparticles were evenly distributed and the particle size was the same.The sedimentation volume ratio was 1,and the re-dispersibility was good.The average particle size was 741.9 nm;Zeta potential was -7.69 mV.The inhibition zone diameters of tilmicosin standard,quercetin-tilmicosin mixture nanoparticles and commercial tilmicosin solution were 2.54,1.51 and 1.38 cm,respectively,and the minimum inhibitory concentrations were 1,1 and 2 μg/mL,respectively.The in vitro killing curves showed that quercetin-tilmicosin mixture nanoparticles against SASCVs strains showed obvious concentration-dependent.【Conclusion】 The quercetin-tilmicosin mixture nanoparticles prepared in this study had strong bactericidal ability against SASCVs strains,and could provide basic research data for the treatment of dairy cow mastitis caused by SASCVs strain.
Cloning,Bioinformatics Analysis and Truncated Fragment Expression for Truncated M Gene of Porcine Epidemic Diarrhea Virus
ZHANG Mingliang, GENG Yachun, LIAN Kaiqi, MA Lei, CHANG Ying, WANG Shuangshan, ZHANG Fuliang
2022, 49(4):  1479-1487.  doi:10.16431/j.cnki.1671-7236.2022.04.030
Abstract ( 172 )   PDF (4958KB) ( 50 )  
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【Objective】 The aim of this study was to investigate the sequence structure and prokaryotic expression of Porcine epidemic diarrhea virus (PEDV) truncated M protein.【Method】 A pair of specific primers were designed according to the published M gene sequence of PEDV.The truncated M gene (rM) of PEDV was amplified by RT-PCR and cloned using RNA extracted from positive clinical samples.The structural characteristics of rM protein were predicted by online bioinformatics software.The obtained truncated M gene was cloned into prokaryotic expression vector pET-28a(+),and the prokaryotic expression plasmid pET-28a-rM was constructed.The correctly identified pET-28a-rM was transformed into E.coli BL21(DE3) competent cells,and the recombinant strain BL21(pET-28a-rM) was successfully constructed,then the recombinant strain was induced by IPTG,the expression conditions of the recombinant protein were optimized,and the recombinant protein was purified by affinity chromatography and detected by SDS-PAGE and Western blotting.Meanwhile,rabbit anti-rM polyclonal antibody was prepared by using the recombinant protein.【Result】 The truncated M gene with a size of 366 bp was cloned.The recombinant protein was composed of 121 amino acids,and the predicted molecular weight was about 12.8 ku.The secondary structure of the protein was composed of random coil,extended strand,beta turn and alpha helix,and the components were 48.76%,33.88%,9.09% and 8.26%,respectively.The protein contained no signal peptide,but had transmembrane region,containing 21 phosphorylation sites.SDS-PAGE results showed that the size of recombinant protein was about 15 ku and existed with the form of inclusion body protein.The protein expression was the highest when induced at 37 ℃ with 1 mmol/L IPTG for 12 h.Western blotting result showed that the recombinant protein had good reactogenicity with the PEDV positive serum.The high immune serum with the titer higher than 1∶51 200 was obtained after the New Zealand White rabbits immunized with purified recombinant protein.【Conclusion】 This study successfully cloned the PEDV truncated M gene the bioinformatics analysis was carried out,and obtained high-purity rM protein,and established a material foundation for the development of biological products for porcine epidemic diarrhea treatment and detection.
Influence of Avian Reticuloendotheliosis Virus Infection on Expression of Bcl-2 and C-myc Genes and Apoptosis in Bursa of Fabricius Cells of SPF Chickens
GU Xianzhe, HOU Ning, LYU Xiaoping, GAO Xueli, LIU Chaonan, WU Wenjie, ZHENG Shimin
2022, 49(4):  1488-1496.  doi:10.16431/j.cnki.1671-7236.2022.04.031
Abstract ( 155 )   PDF (9128KB) ( 29 )  
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【Objective】 This experiment was aimed to investigate the mechanism of apoptosis of immune organs caused by avian Reticuloendotheliosis virus (REV).【Method】 SPF chickens were selected as experimental subjects in this study.100 1-day-old SPF chickens were randomly divided into REV-infected group and uninfected control group.Those REV-infected chickens were taken 500 μL of REV dilution by peritoneal injection,the control group chicks were injected with sterilized saline instead.On the 1st,7th,14th,21th,28th and 42th day after virus infection,5 chickens were randomly selected from these two groups of chickens,and their bursas were quickly removed after execution.HE staining and pathological imaging system were applied to observe the nuclear-cytoplasm ratio of bursa of Fabricius cells,TUNEL assay was used to determine the quantity of apoptotic cells,immunohistochemical method was used to count the number of Bcl-2 and C-myc positive cells,and Real-time PCR and ELISA methods were used to test the mRNA expression and protein content of Bcl-2 and C-myc genes in the bursa of Fabricius.【Result】 The results showed that:①21-42 days after SPF chickens infected with REV,the percentage of apoptosis of bursal lymphocytes was significantly or extremely significantly higher than that of the control chickens (P<0.05;P<0.01);②21-28 days after SPF chickens infected with REV,the ratio of lymphocyte to cytoplasm in the bursa of Fabricius was significantly lower than that of control chickens (P<0.05);③The number of Bcl-2 and C-myc positive cells in the bursa of Fabricius of SPF chickens infected with REV was significantly higher than that of control chickens at 21 and 28 d (P<0.05);④21 d after REV infection,the expression of Bcl-2 and C-myc genes mRNA in bursa of Fabricius was extremely significantly higher than that of control chickens (P<0.01);⑤After SPF chickens infected with REV,the Bcl-2 protein content in the bursa of Fabricius was increased to different degrees compared with control group chickens,and the difference between 21 and 28 d was extremely significant (P<0.01) and significant (P<0.05),respectively.The content of C-myc protein in the REV-infected group was always higher than that in the control group,and it was increased extremely significantly from 21 to 28 d (P<0.01).【Conclusion】 The above results indicated that the abnormal expression of Bcl-2 and C-myc in the bursa of Fabricius of SPF chickens caused by REV infection was related to the apoptosis of bursal of Fabricius cells caused by the virus,and the increased apoptosis in the number of bursal of Fabricius cell was related to the suppression of immune function caused by REV infection.
Isolation,Identification of Duck Tembusu Virus,and Genetic and Evolutionary Analysis of Its Envelope Protein
YIN Yajie, LIANG Ruiying, CUI Huan, MENG Lijia, NI Weiling, QIAO Sina, GUO Kangkang, ZHANG Bo, LI Songli, HOU Shaohua, DONG Shishan
2022, 49(4):  1497-1506.  doi:10.16431/j.cnki.1671-7236.2022.04.032
Abstract ( 197 )   PDF (10345KB) ( 51 )  
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【Objective】 The aim of this study was to understand and grasp the epidemiological and biological characteristics of Duck Tembusu virus (DTMUV),in order to provide theoretical basis and technical support for DTMUV prevention and treatment.【Method】 The pathogen was isolated from 10 ducks showing typical clinical signs of DTMUV from duck farm in Hebei province by cell and chicken embryo inoculation method,and was identified by RT-PCR,transmission electron microscopy,Western blotting and indirect immunofluorescence assay (IFA).The animal regression test was performed to detect its virulency,and the inheritance of envelop protein was carried out.【Result】 The isolated DTMUV strain could well proliferate in DF-1 cells and produce typical cytopathic effect (CPE) and had ability to kill the chicken embryo.After purification,the virus particles with a diameter of 30-60 nm were observed under electron microscopy.RT-PCR results showed that there was a single band at about 270 bp, which was consistent with the expected size of DTMUV; Western blotting results showed that there was a specific band at 60 ku, which was consistent with the size of E protein; The results of IFA showed that after DF-1 cells were inoculated with virus, bright specific fluorescence appeared in the cytoplasm, the above results showed that the isolated virus was DTMUV.The identified virus strain was named as AX2020 strain.After intramuscular injection,the infection rate of AX2020 strain infected Peking duck was as high as 100%,the diseased ducks had typical clinical symptoms such as neurological symptoms and diarrhea.Through sequence alignment,it was found that AX2020 strain had the highest similarity with GA strain (MK907880.1),and was far away from SD14 strain (MH748542.1).Compared with commercial inactivated vaccine strain HB2010 (MN649262.1) and live vaccine strain FX2010 (MH414568.1),amino acid site mutations had occurred at positions 93,277 and 487 of AX2020 strain.【Conclusion】 A DTMUV strain AX2020 was successfully isolated,which showed strong pathogenicity in Peking ducks.Compared with the domestic vaccine strains,the envelope protein of AX2020 strain had mutation at amino acid site,which laid a certain foundation for epidemiological investigations and vaccine-related studies.
Research Progress on Newcastle Disease Virus-like Particles
CHI Shanshan, JIA Weijuan, HE Yunjiang, MENG Qinglei, CHEN Yunjiao, WANG Xueli
2022, 49(4):  1507-1515.  doi:10.16431/j.cnki.1671-7236.2022.04.033
Abstract ( 297 )   PDF (2802KB) ( 134 )  
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Newcastle disease (ND) is an acute,highly contagious avian infectious disease caused by Newcastle disease virus (NDV),with respiratory,digestive and nervous system symptoms as the main characteristics,the mortality rate in susceptible poultry is as high as 100%,which has brought serious economic losses to poultry breeding and trade in China and the world.Virus-like particles (VLPs) are virus-like protein particles that are formed by in vitro expression or spontaneous assembly of viral proteins during the infection process,which do not contain viral genome,cannot replicate,and have no infectious ability.The suitable size and unique surface structure of VLPs can be effectively recognized by natural immune cells and molecules,and induce good innate and adaptive immune responses.In addition,VLPs do not contain viral infectious nucleic acids and are highly safe.In recent years,they have become a research hotspot in the field of vaccines.Dendritic cells (DCs),as full-time antigen-presenting cells,have a unique function between innate immunity and adaptive immunity,and are currently the full-time immune cells with the strongest antigen-presenting ability.Migration of mature DCs (mDCs) to secondary lymphoid tissues can stimulate the activation and proliferation of primary T cells,and is considered to be the initiator of specific immune responses.Immature DCs (imDCs) cultured in vitro can also help enhance the ability of antigen presentation.NDV-VLPs mainly use NDV-M protein as the backbone to assemble HN,F and NP proteins,which can show similar appearance and immunogenicity to live NDV particles.It can be seen that vaccines made by NDV-VLPs also have many advantages,such as safety,stability,stimulating humoral and cellular immune responses,expressing other antigens as carriers,and being designed to distinguish natural infections from immunization.Therefore,the article mainly discusses the related content of NDV-VLPs in order to provide reference for follow-up research.
Isolation and Identification of Mycoplasma synoviae Guizhou Strain and Sequence Analysis of VlhA Gene
SONG Chun, WANG Bolin, LI Mei, WEN Ming, WANG Kaigong, CHEN Changxiu, CHENG Zhentao
2022, 49(4):  1516-1523.  doi:10.16431/j.cnki.1671-7236.2022.04.034
Abstract ( 160 )   PDF (2015KB) ( 28 )  
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【Objective】 The aim of this study was to obtain Mycoplasma synoviae(MS) strains in Guizhou,to further develop its etiology and immunology related research.【Method】 A total of 107 samples of pharyngeal swabs,swollen joints and claw pads and swollen tissue exudate suspected to be infect with MS were collected from a broiler farm in Zhijin county,Guizhou province and detected by PCR.The samples tested positive by PCR were used to isolate and culture MS,Wright staining,biochemical test and sequence analysis of the VlhA gene were performed.【Result】 20 positive samples were tested by PCR,only 1 strain of MS was isolated from the positive samples of joint and claw pad,and the colony formed "egg-shaped" on solid medium.The isolated strain was small blue spherical stained by Wright’s in the microscope.In biochemical tests,the isolates could decompose glucose and maltose,but not arginine and urea,which was consistent with the biochemical results of MS standard strains.The target fragment of 821 bp was amplified by PCR.Nucleotide similarity analysis of the VlhA gene of the isolated strain indicated that the strain was MS,and there were regional differences in similarity with other endemic strains in GenBank.The similarity with the epidemic strains in Anhui,Chongqing,Fujian,Guangdong,Guangxi,Hubei,Hunan,Jiangsu and Yunnan was 90.3% to 99.7%.The most similar strain was MS epidemic strain in Hunan,which reached 99.7%.But the similarity with foreign reference strains was low.The phylogenetic analysis of VlhA gene showed that this strain was closely related to the domestic reference strain,but far from the foreign.【Conclusion】 In this study,a Guizhou endemic MS strain was successfully isolated and identified from chickens causing swelling of joints and foot pads with some variation.This study provided a basis for the molecular and biological characteristics of MS in Guizhou and the diagnosis and control of this disease.
Establishment of PMA-PCR Method for Detection of Orf Virus
LIANG Qian, BAO Taotao, XIAN Simei, YANG Qian, LI Pengfei, GU Qinglin, ZHENG Weihao, DU Peng, QING Chengxin
2022, 49(4):  1524-1531.  doi:10.16431/j.cnki.1671-7236.2022.04.035
Abstract ( 210 )   PDF (4356KB) ( 32 )  
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【Objective】 In order to quickly and effectively evaluate whether Orf virus (OrfV) in the host animal’s body and surrounding environments were pathogenic,the nucleic acid dye propidium bromide (PMA) combined with PCR amplification technology was used to target F1L gene,so as to establish a PMA-PCR detection method for OrfV with infectious activity.【Method】 The virus suspension was treated with water bath at different temperatures for the same time,and the optimal heat inactivation temperature of virus samples was determined by PCR.The exposure time gradient and concentration gradient of PMA were set respectively,and the exposure time and working concentration of PMA were further optimized to determine the best treatment conditions for PMA to effectively distinguish between infectious active OrfV and inactivated OrfV.The specificity of the established PMA-PCR method was verified by specificity test,and the sensitivity of the method was verified by detecting different proportion of heat inactivated OrfV and active OrfV mixed virus suspension samples.【Result】 OrfV samples could be completely inactivated after 10 min of treatment in a hot water bath at 60 ℃.PMA was covalently bound to the DNA of inactivated OrfV and the best exposure time for photolysis of free PMA in the solution was 15 min.The minimum PMA concentration at which the amplification of heat inactivated OrfV genome was completely inhibited was 20 μmol/L,the maximum PMA concentration of uninhibited amplification of active OrfV genome was 35 μmol/L.Specific test result showed that this method had a positive band when detecting active OrfV.The amplification results of the FMDV,SPPV and GTPV groups were all negative.After PMA treatment,the sensitivity of detecting active OrfV in different ratios of live and heat-inactivated OrfV suspensions was 10-1.68 TCID50/0.1 mL.【Conclusion】 This experiment had successfully established a PMA-PCR detection technology for active OrfV,which had strong specificity and high sensitivity,and could provide technical reference for evaluating the pathogenicity of OrfV in related work areas.
Basic Veterinary Medicine
Study on Acute and Subchronic Toxicity of Polygonum hydropiper L. Extract
ZHANG Wen, QIN Zhibiao, CHENG Jing, YU Meiling, WEI Yingyi, LI Jiang, HU Tingjun
2022, 49(4):  1532-1544.  doi:10.16431/j.cnki.1671-7236.2022.04.036
Abstract ( 190 )   PDF (10389KB) ( 32 )  
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【Objective】 The aim of this study was to determine the toxicity and safety of Polygonum hydropiper L. extract.【Method】 In the acute toxicity test,20 healthy SPF Kunming mice were randomly divided into 6 groups:Control group 0 g/kg BW and five treatment groups with Polygonum hydropiper L. extract at doses of 20,10,5,2.5 and 1.25 g/kg BW.The toxicity and death of the animals were observed for 7 days.In the maximum dose test of acute toxicity test,20 mice were given the drug by equal volume for 3 times within 24 h,and their physiological status,poisoning and death were observed.On the 8th day post treatment,their organs were observed for abnormalities.In the 30-day feeding experiment,80 healthy SPF SD rats were randomly divided into 4 groups:Control group 0 g/kg BW and Polygonum hydropiper L. high,medium and low dose groups (20,10 and 5 g/kg BW) with 20 mice each group (half female and half male).The rats were treated for 30 days.Their weight,food intake and water intake were recorded daily.On the 7th day after drug withdrawal,blood samples were collected for routine blood test and blood biochemistry test,organ index was calculated,and histopathological examination was performed.【Result】 The results showed that no death occurred in all groups in acute toxicity test,and the LD50 of Polygonum hydropiper L. extract was not obtained.The maximum tolerance of Polygonum hydropiper L. extract in mice was 30 g/kg BW,indicating that the Polygonum hydropiper L. extract was safe and had no acute toxicity.In the subchronic toxicity test,although the body weight,water intake,feed intake and organ index of rats in each administration group were different from those of the control group,they were within the normal range.The white blood cell and lymphocyte indexes of the rats in high dose group decreased significantly (P<0.05),and the neutrophils increased significantly (P<0.05).The white blood cell index of the rats in the middle dose group decreased significantly (P<0.05;P<0.01),there was no significant difference among the other groups (P>0.05).Among the blood biochemical indexes,compared with control group,the TG level of rats in high dose group increased significantly (P<0.05),and the levels of BUN,CREA and TBIL decreased significantly (P<0.05).The BUN level of rats in middle dose group decreased significantly (P<0.05).CREA levels increased significantly (P<0.05),ALT and TG levels in low dose group increased significantly (P<0.05),BUN levels decreased significantly (P<0.05),and there was no significant difference between the other groups (P>0.05).Necropsy and pathological examination showed no obvious abnormal changes,indicating that the extract of Polygonum hydropiper L. was safe and had no subchronic toxicity.【Conclusion】 In brief, Polygonum hydropiper L. spp. extract of 5 g/kg BW and below was safe and nontoxic.
Study on the Mechanism of Traditional Chinese Medicine Compound in the Treatment of Endometritis in Dairy Cows Based on Network Pharmacology
WANG Yuefu, XU Chuang, CUI Yizhe
2022, 49(4):  1545-1555.  doi:10.16431/j.cnki.1671-7236.2022.04.037
Abstract ( 205 )   PDF (8647KB) ( 78 )  
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【Objective】 The traditional Chinese medicine compound consisting of Salvia miltiorrhiza,Leonurus heterophyllus, Scutellaria baicalensis and Forsythia suspensa was analyzed by network pharmacology,in order to study the possible targets of its effective components for treating dairy cow endometritis,and explain the mechanism of target action pathway.【Method】 TCMSP database was used to screen the target of drug components,NCBI,GeneCard,geneMap,TTD and DrugBank were used to find the related targets of cow endometritis,and the common gene targets of drug components and cow endometritis were screened out,which were used as the predicted targets for the treatment of cow endometritis with this traditional Chinese medicine compound,and then uploaded to STRING database to establish PPI network,which was screened by Cytoscape 3.8.2 software according to the connectivity.Metascape database was used to analyze the enrichment of GO function and KEGG pathway of core targets,and its mechanism was explored.【Result】 133 kinds of effective active ingredients were screened out from the traditional Chinese medicine compound,mainly including quercetin,kaempferol,baicalein,tanshinone ⅡA,etc.,292 corresponding target proteins were found through drug ingredients,130 related targets of cow endometritis were found through database,and 24 predicted targets of traditional Chinese medicine compound for treating cow endometritis were screened out,among which 7 core targets were screened out,including tumor necrosis factor,protein kinase and prostaglandin-endoperoxide synthase 2.GO function enrichment analysis resulted in 20 items of biological process,6 items of molecular function and 6 items of cell component.Biological processes included the reaction of lipopolysaccharide,the positive regulation of leukocyte-cell adhesion,the reaction to inorganic substances,etc.Molecular function included signal receptor activity regulation,transcription coactivator binding,heme binding and so on.Cell components included cell membrane raft,membrane side,granular secretory cavity,etc.The enrichment analysis of KEGG pathway involved 20 genes,and there were 9 pathways involved in regulation.Among them,many genes included AGE-RAGE signaling pathway,cancer proteoglycan pathway,blood flow shear stress and atherosclerosis pathway and Th17 cell differentiation pathway.【Conclusion】 The traditional Chinese medicine compound might have therapeutic effect on cow endometritis by regulating tumor necrosis factor,protein kinase,prostaglandin-endoperoxide synthase 2 and other targets,and by participating in AGE-RAGE signaling pathway and Th17 cell differentiation.
Isolation and Identification of Skin Pathogenic Bacteria from Dogs and Cats and Analysis of Antibacterial Effect of Natural Products
ZHAO Tianrui, SHAO Jinhua, WANG Chaohao, YAN Rui, LEI Yifei, LIU Xiaoya, HU Changmin
2022, 49(4):  1556-1567.  doi:10.16431/j.cnki.1671-7236.2022.04.038
Abstract ( 288 )   PDF (6821KB) ( 78 )  
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【Objective】 The purpose of the experiment was to isolate and identify the bacterial pathogens of dogs and cats suffering from skin diseases in Wuhan,and to explore their sensitivity to traditional antibiotics and natural active products gambogic acid (GA) and 6-bromoindirubin-3’-oxime (BIO).【Method】 Skin pathogen bacteria from dogs and cats were collected and isolated,as well as growth characteristics,Gram staining microscope observation and PCR were determined.SPF mice were used to verify pathogenicity.The drug resistance to traditional drugs was verified by drug sensitive paper,and the minimum inhibitory concentration (MIC) of natural products was measured.【Result】 2 strains of Staphylococcus aureus,3 strains of Staphylococcus pseudointermediate,2 strains of Staphylococcus felis,1 strain of Streptococcus canis,and 1 strain of Proteus mirabillis were isolated.The pathogenicity was verified by skin infection in SPF mice.Streptococcus canis and Proteus mirabillis were sensitive to the tested drugs.Staphylococcus aureus had different resistance to cotrimoxazole,penicillin,erythromycin,tetracycline,levofloxacin,oxacillin,gentamicin,clindamycin and chloramphenicol.The natural active products of GA and BIO had good antibacterial effects on the pathogenic bacteria,and except strain F5,the MIC of GA was universally lower than that of BIO.【Conclusion】 In the study,a total of 9 pathogenic bacteria were isolated from dogs and cats.Streptococcus canis and Proteus mirabilis were sensitive to traditional antimicrobial agents,and some Staphylococcus strains were resistant to them.GA and BIO had obvious antibacterial effects against skin pathogenic bacteria.It showed that GA and BIO could be used as potential antibacterial drugs to prevent and control skin bacterial disease in dogs and cats.
Effect of Chuanwang Xiaoyan Powder on LPS Induced Acute Lung Injury in Rats
WANG Lu, HU Panpan, CHENG Jia, SUN Panpan, SUN Na, FAN Kuohai, YIN Wei, LI Hongquan, SUN Yaogui
2022, 49(4):  1568-1576.  doi:10.16431/j.cnki.1671-7236.2022.04.039
Abstract ( 288 )   PDF (3452KB) ( 50 )  
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【Objective】 The experiment was conducted to study the therapeutic effect of Chuanwang Xiaoyan powder on lipopolysaccharide (LPS) induced acute lung injury in rats.【Methods】 60 SD male rats were randomly divided into blank group,levofloxacin-positive drug group,LPS model group,low-dose (1 g/kg BW),medium-dose (2 g/kg BW) and high-dose (4 g/kg BW) Chuanwang Xiaoyan powder groups.Each group contains 10 rats.A rat model of acute lung injury was made with 3 mg/kg LPS in the model group and the administration groups by the nasal drip method for 24 h.After successful modeling,the drug administration groups were given the corresponding doses of drugs by gavage,and the model and blank groups were given the same doses of saline by gavage.After 4 d of treatment,the rats were sacrificed,the serum was separated,and the lung tissues were fixed and cryopreserved.The wet/dry weight ratio of rat lung tissue was detected in each group.HE staining was used to observe the pathological changes of rat lung tissue.ELISA method was used to detect the content of IL-1β and IL-6 in serum,and the mRNA and protein expression levels of IL-1β and IL-6 in the lung tissue were respectively detected by Real-time quantitative PCR and Western blotting.【Results】 Compared with the blank group,rats in the LPS model group had thicker lung interstitial walls,infiltration of inflammatory cells in alveolar cavity,bleeding,and destruction of alveolar structure and other symptoms of pneumonia.The wet/dry weight ratio of lung tissue was increased extremely (P<0.01).The levels of IL-1β and IL-6 in serum,and the mRNA expression levels and protein expression levels of IL-1β and IL-6 in lung tissues were significantly increased (P<0.05).Compared with the LPS model group,the pulmonary interstitial thickening of the rats in the drug treatment groups was alleviated.The wet/dry weight ratio of lung tissue,the expression levels of inflammatory factors IL-1β,IL-6 mRNA and protein expressions in serum and lung tissue were significantly reduced (P<0.05).【Conclusions】 Chuanwang Xiaoyan powder could effectively inhibit LPS induced acute lung injury and inflammation.
Devolopment of Surface-enhanced Raman Spectroscopy Combined with Immunochromatography for Detecting Cephalexin
CHEN Yan, XUE Qiang, JIN Yong, ZOU Mingqiang, QI Xiaohua, LI Boyi, YIN Hong
2022, 49(4):  1577-1584.  doi:10.16431/j.cnki.1671-7236.2022.04.040
Abstract ( 227 )   PDF (6176KB) ( 35 )  
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【Objective】 This study was aimed to establish a rapid,sensitive,specific and easy-to-use surface-enhanced Raman spectroscopy (SERS) combined with immunochromatographic assay for detecting cephalexin.【Method】 In this study,Raman molecule 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) and cephalexin antibody were conjugated with Au (core)@Ag (shell) nanoparticle to serve as SERS nanoprobe.The gold nanoparticles and Au (core)@Ag (shell) nanoparticles were identified by transmission electron microscopy(TEM) and ultraviolet spectrophotometer.Using the combination of SERS and immunochromatography,the quantitative detection technology of cephalexin was established,and applied to the detection of milk samples.【Result】 The results of TEM and ultraviolet spectrophotometer showed that silver nanoparticles were successfully coated on the surface of gold particles.Raman spectrometer was used to detect the characteristic peaks of Raman signal molecules on the immunochromatographic strip,and the peak height was selected to determine the content of cephalexin.The molecular characteristic peaks of Raman signal were 1 062,1 154,1 334 and 1 558 cm-1,of which the characteristic peak signal at 1 334 cm-1 was the strongest.The peak height was selected to determine the content of cephalexin.The detection linear range was 0 to 1 ng/mL,the detection limit was 0.001 ng/mL,and the half inhibitory concentration was 0.05 ng/mL.The cross-reactivity with cefaclor was 111.1%,with ceftriaxone sodium,cefuroxime axetil and cephalotin sodium were all less than 0.1%,respectively.Recoveries of cephalexin from spiked milk at levels of 1,4 and 8 ng/mL were 94.4%,103.3% and 97.5%,respectively.【Conclusion】 The method of surface-enhanced Raman spectroscopy combined with immunochromatography for detecting cephalexin developed in this study was simple,rapid,sensitive and low-cost,which could be used for on-site mass sample screening and testing.
Mechanism Study of Protocatechuic Acid on LPS-induced Endometritis in Mice
ZHANG Yuzhu, YU Chao, ZHENG Huihua, TANG Xinyue, HUANG Ronglei, XIE Guanghong
2022, 49(4):  1585-1591.  doi:10.16431/j.cnki.1671-7236.2022.04.041
Abstract ( 231 )   PDF (2531KB) ( 87 )  
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【Objective】 This study was aimed to investigate the protective effect and mechanism of protocatechuic acid on lipopolysaccharide (LPS) induced endometritis in mice.【Method】 Sixty mice were randomly divided into control group,LPS group and LPS+ procatechuic acid (20,40,80 mg/kg) groups,with 12 mice in each group.The control group was injected with 50 mL normal saline via microsyringe,the LPS group was injected with 50 mL LPS (1 mg/kg),and the protocatechuic acid treatment groups were injected intraperitoneally with 20,40 and 80 mg/kg protocatechuic acid 1 h before the injection of 50 mL LPS.After 24 h of injection,all the mice were sacrificed by cervical dislocation.The uterus tissues were collected,and the pathological changes of the uterus tissues were detected by HE staining,myeloperoxidase (MPO) activity was detected by kit,and the contents of inflammatory factors tumor necrosis factor α(TNF-α),interleukin 1β(IL-1β) and IL-6 were detected by ELISA.The expression levels of p65 and NF-κB inhibitor protein (IκB) and phosphorylaed p65 and IκB protein (p-p65 and p-IκB) were detected by Western blotting.【Result】 Histopathological results showed that the endometrial epithelial structure of mice in control group was normal.The uterus tissues of mice in LPS group showed severe inflammatory cell infiltration and endometrial epithelial hyperemia and edema.Compared with LPS group,with the increase of protocatechuic acid concentration,the inflammatory cells in the uterus tissues of mice were significantly reduced,and the hyperemia and edema of endometrial epithelium were significantly improved.The activity of MPO in LPS group was extremely significantly higher than that in control group (P<0.01).Compared with LPS group,MPO activity in protocatechuic acid treatment group was extremely significantly decreased in a dose-dependent manner (P<0.01).ELISA results showed that compared with control group,the contents of TNF-α,IL-1β and IL-6 in endometrial tissues of mice in LPS group were extremely significantly increased (P<0.01).Compared with LPS group,the contents of inflammatory cytokines TNF-α,IL-1β and IL-6 in endometrial tissues of mice treated with protocatechuic acid were extremely significantly decreased (P<0.01) in a dose-dependent manner.Western blotting results showed that the expression of p-p65 and p-IκB in LPS group were extremely significantly increased compared with control group (P<0.01).Compared with LPS group,the expression of p-p65 and p-IκB in protocatechuic acid treatment group were extremely significantly decreased in a dose-dependent manner (P<0.01).【Conclusion】 Protocatechuic acid played a protective effect on lPS-induced endometritis in mice by alleviating pathological changes,decreasing MPO activity,inhibiting NF-κB signaling pathway and the production of inflammatory cytokines.
Infection Status and Molecular Characterization of Giardia duodenalis in Pigs in Guangdong Province
ZHONG Menglong, ZHANG Haoji, CHEN Xiangjie, LI Fukun, QIU Xixi, LI Yi, HUANG Fuqiang, WANG Nina, YU Xingang
2022, 49(4):  1592-1598.  doi:10.16431/j.cnki.1671-7236.2022.04.042
Abstract ( 212 )   PDF (2207KB) ( 162 )  
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【Objective】 This study was aimed to evaluate the zoonotic risk of Giardia duodenalis (G.duodenalis) in pigs,and investgate the prevalence and molecular characterization of G.duodenalis in different citys of Guangdong province.【Method】 Fresh pig feces samples were collected from 10 regions in Guangdong province and examined by microscope.Nested PCR was used to amplify the glutamate dehydrogenase (gdh) gene of G.duodenalis,and the positive rates of G.duodenalis in different regions and growth stages were calculated according to the amplification results.All PCR positive products were sequenced,and the gdh gene sequences of G.duodenalis were BLAST compared to determine the genotype of G.duodenalis.The maximum likelihood method of Mega 7.0 software was used to construct the evolutionary tree.【Result】 The cyst of G.duodenalis was oval with thick wall and the size was (10-14) μm×(7-10) μm.It had an obvious longitudinal axis column,and the nucleus was distributed on both sides of the axis column.94 positive samples were detected from 521 samples,and the average positive rate was 18.04%(94/521),among which Maoming (40.00%,10/25) and Qingyuan (35.06%,27/77) had higher positive rate,but Zhaoqing and Shaoguan were lower,with 3.90%(3/77) and 8.57%(3/35) respectively.The positive rates in Foshan,Jiangmen,Yangjiang,Heyuan,Guangzhou and Huizhou were 11.11%(2/18),15.31%(15/98),11.43%(4/35),13.33%(4/30),9.38%(3/32) and 24.47%(23/94),respectively.The positive rate of G.duodenalis in sows (24.55%) was significantly higher than that in weaned piglets (13.30%)(P<0.05),but there was no significant difference between the positive rate of G.duodenalis in sows and fattening pigs (19.23%)(P>0.05).Based on genotyping and phylogenetic analysis of gdh gene,a total of 5 G.duodenalis subtypes were found,including assemblage AⅠ and assemblage E1,E2,E10 and E13,of which 61.70% (58/94) G.duodenalis positive samples belonged to sub-assemblage AⅠ and 38.30% (36/94) belonged to assemblage E.【Conclusion】 The infection of G.duodenalis from pigs in Guangdong province was common,and the positive rates of pigs in different regions and different growth stages were different.The zoonotic sub-assemblage AⅠ was the dominant sub-assemblage,indicating that there was a risk of transmission from animals to humans or vice versa and the possible public health problems caused by the G.duodenalis should be taken seriously.