【Objective】 The purpose of this study was to clone Luman/CREB3 recruitment factor(CREBRF) gene of Ovis aries and analyze its bioinformatics,and detect the expression of CREBRF gene in different tissues of Ovis aries,so as to provide theoretical reference for exploring the biological function of CREBRF gene in Ovis aries.【Methods】 Using ovary cDNA of Ovis aries as template,the complete CDS region of CREBRF gene was amplified by PCR and cloned,the similarity alignment,phylogenetic tree and bioinformatics analysis were carried out.The expression of CREBRF gene in different tissues of Ovis aries was detected by Real-time quantitative PCR.【Result】 The total length of CDS region of CREBRF gene was 1 920 bp,encoding 639 amino acids.The similarity alignment results showed that the similarity of CREBRF amino acid sequence of Ovis aries with Capra hircus,Bos taurus,Homo sapiens,Mus musculus,Sus scrofas,Canis lupus familiaris,Equus przewalskii,Gallus gallus,Anas platyrhynchos and Danio rerio were 99.8%,99.1%,95.4%,93.6%,98.3%,97.5%,98.4%,88.0%,87.5% and 61.3%,respectively.The phylogenetic tree analysis showed that Ovis aries had the closest relationship with Capra hircus and Bos taurus,and the furthest relationship with Danio rerio.Bioinformatics analysis found that the molecular formula of CREBRF protein in Ovis aries was C₃₁₂₆H₄₉₁₄N₈₅₈O₁₀₅₆S₂₁,the theoretical molecular weight was 72.08 ku,the theoretical isoelectric point (pI) was 4.77,the half-life was 30 h,the N-terminal of peptide chain was methionine (Met),and the instability coefficient was 54.83.CREBRF protein existed in the nucleus,there was no transmembrane property and signal peptide,and was a hydrophilic unstable protein.The secondary structure of CREBRF protein was mainly random random coil (47.57%),followed by alpha helix(37.09%).Real-time quantitative PCR results showed that CREBRF gene was expressed in different tissues of Ovis aries,the expression in heart,kidney and ovary were significantly higher than that in other tissues (P<0.05).【Conclusion】 This experiment obtained the full-length sequence of CREBRF gene,and preliminarily studied its tissue expression,which provided materials for studying the regulatory mechanism of embryonic development and improving fecundity in Ovis aries.

【Objective】 In order to obtain the full-length transcriptome database of abomasum in yak (Bos grunniens),the functional genes of abomasum of yak were deeply excavated.【Method】 The full-length transcriptome of abomasum of adult yak was sequenced by single molecular real time (SMRT) sequencing technology using high-throughput sequencing PacBio Sequel high-throughput sequencing system.The original data were quality controlled and de redundant analysis,and then compared with the reference genome to obtain filtered non repetitive sequences (Unigenes).A variety of bioinformatics software were used to compare the full-length transcriptome data of abomasum in yak for functional annotation,transcription factor annotation,coding region prediction,simple sequence repeats (SSR) analysis and alternative splices analysis.【Result】 A total of 14 467 420 subsequences were obtained by sequencing,with an average length of 3 344.23 bp.296 840 CCS sequences and 277 402 FLNC sequences were obtained by quality control.After filtering and de redundancy,the reference genome was compared,and 8 556 Unigenes were finally obtained.By comparing with NR,Swiss-Prot,KEGG,KOG,eggNOG,GO and Pfam databases,Unigenes were annotated,of which 8 544 Unigenes were annotated in NR database.Swiss-Prot database annotated 8 475 Unigenes.The KEGG database annotated 1 721 unigenes.The KOG database annotated 6 572 Unigenes.The eggNOG database annotated 8 491 Unigenes.GO database annotated 7 725 Unigenes;The Pfam database annotated 8 162 Unigenes.In addition,943 transcription factors,8 544 CDS fragments,3 596 SSR sites and 1 825 alternative splicing events were identified or predicted.【Conclusion】 In this study,reliable full-length transcriptome data of yak abomasum were obtained,it could provide data support for the further study of the biological characteristics,related metabolic pathways,signal pathways and molecular mechanisms of abomasum in yak.

【Objective】 The purpose of this study was to obtain LbCas12a protein from Lachnospiraceae ND2006 with in vitro cleavage activity,in order to provide an important biological tool for the application of LbCas12a.【Method】 LbCas12a gene was synthesized according to the gene sequence of pMBP-LbCas12a plasmid (Addgene,113431),and homologous recombination was performed with pET-28a(+) linearized vector.The recombinant plasmid pET28a-LbCas12a was constructed.After sequencing and double enzyme digestion,the positive recombinant plasmid was transferred into the BL21(DE3) for IPTG induced expression.The optimal concentration and temperature induced by IPTG were detected by 12% SDS-PAGE,and the expression form was identified.The protein concentration was detected by Ni-NTA resin affinity chromatography and ultrafiltration concentration,and the protein concentration was detected by BCA method.The concentrated LbCas12a was co-incubated with Porcine parvovirus (PPV) target DNA,CRISPR RNA (crRNA) and ssDNA reporter probe FQ,a control group without LbCas12a protein and four experimental groups with different protein concentration gradients (125,250,500,1 000 nmol/L) were set up.The fluorescence intensity of ssDNA probes in different groups was detected by automatic microplate reader,and the activity of PPV at the test site was detected.【Results】 Sequencing and NheⅠ and SalⅠ double enzyme digestion results showed that the recombinant plasmid pET28a-LbCas12a was successfully constructed.The results of 12% SDS-PAGE showed that the optimum induction concentration of IPTG was 0.5 mmol/L,the optimum induction temperature was 37 ℃,and the expression form was mainly soluble expression.The results of protein concentration detection showed that the concentration of protein was 485 ng/μL and the weight was 143 ku.The activity test results showed that the fluorescence intensity of 125,250,500,1 000 nmol/L LbCas12a protein was extremely significantly higher than that of control group (P<0.01).【Conclusion】 In this study,LbCas12a protein with high activity was successfully expressed,and LbCas12a protein had trans-cleavage activity of ssDNA in vitro,which laid a foundation for the subsequent molecular detection technology based on CRISPR-LbCas12a system.

【Objective】 The purpose of this study was to construct the multi copper oxidase (BMCO) gene deletion strain of Brucella bovis S2308,explore the growth characteristics of the deletion strain and its viability in host cells,and analyze the structure of BMCO protein.【Method】 The upstream and downstream homologous arms of BMCO gene and Kan gene were amplified by PCR,and the three genes were fused by fusion PCR.The fusion fragment was connected with pMD19-T vector to make Brucella bovis S2308 competent cells.1 800 V voltage and 400 Ω resistance were transferred to Brucella bovis S2308 competent cells and coated in Kan-resistant Brucella solid medium.Positive colonies were selected and cultured continuously for 10 generations.The positive genetic stability and growth trend of the 10th generation positive colonies were detected.The pBBR1MCS-4-BMCO fusion plasmid was electrotransferred into Brucella bovis which could stably inherit BMCO gene deletion,and the positive colonies were cultured and screened.The mouse macrophage RAW264.7 was infected with parent strain,deletion strain and supplementary strain with plural infection with MOI 100.The viability of the three strains in the cells was detected by plate counting method.【Result】 The results showed that the upstream and downstream homologous arms of BMCO gene and Kan gene with fragment size of 522,539 and 1 054 bp were successfully obtained,the recombinant vector of pMD19-T-BMCO-Kan fusion fragment was successfully constructed,the stable genetic deletion strain of BMCO gene was obtained,named S2308ΔBMCO,and the complementary strain of BMCO gene was successfully constructed,named ΔBMCO::BMCOS2308.The growth curve of the deleted strain was the same as that of the parent strain,which reached logarithmic growth phase at 12 h and entered the plateau phase at 30 h.After infecting mouse macrophage RWA264.7,compared with the parent strain S2308,the viability of the S2308ΔBMCO was extremely significantly decreased (P<0.01).Bioinformatics analysis showed that BMCO was a hydrophobic protein,located in the cytoplasm and contained a large number of random coil,alpha helix and extended chain,indicating that the protein had multiple binding sites.【Conclusion】 The deletion and replacement strains of BMCO gene of Brucella were successfully constructed.The deletion of BMCO gene did not affect its growth performance,but its viability in host cells decreased significantly.The structure of BMCO protein was preliminarily analyzed,which would lay a foundation for the further study of the function of protein secreted by Brucella.

【Objective】 The aim of this study was to clone Msh-homeobox 2 (MSX2) gene in chickens,and analyze its bioinformatics and embryonic expression patterns,so as to provide support for further study on the structure and function of MSX2 gene.【Method】 The 80 Langya chicken eggs were incubated,the samples were collected on 1st,2nd,3rd,4th,5th,6th,9th,12th,15th and 18th days of incubation,the whole embryos were collected from 1 to 6 embryonic age,the heart and liver were collected from 9,12,15 and 18 embryonic age,respectively).Total RNA was extracted,MSX2 gene was amplified by RT-PCR and cloned,bioinformatics analysis was carried out.Real-time quantitative PCR was used to analyze its expression level in embryo and tissues of Langya chickens.【Result】 MSX2 gene was successfully cloned from Langya chicken embryo with a sequence length of 818 bp,and an open reading frame of 780 bp,encoding 259 amino acids.The MSX2 amino acid sequence of Langya chickens was 99.23% with that of Coturnix japonica,Haliaeetus leucocephalus and Tyto alba alba,and the lowest with that of Capra hircus,which was 74.54%.The phylogenetic tree analysis results showed that Langya chickens and Coturnix japonica was clustered together.Bioinformatics analysis showed that the molecular weight of MSX2 gene was 28 235.18 u,and the isoelectric point (pI) was 9.71.MSX2 protein had no signal peptide and transmembrane domain,and were unstable hydrophilic proteins,and the majority of MSX2 protein was distributed in nucleus (60.9%).The MSX2 protein had 37 phosphorylation sites,8 O-glycosylation sites and 1 N-glycosylation site.The secondary structure of MSX2 protein was mainly composed of random coil (63.32%) and alpha helix (28.19%).Real-time quantitative PCR analysis showed that the expression of MSX2 gene in whole embryo (1-6 embryonic ages) were firstly increased and then decreased,and the expression were the highest at 4 embryonic age,and was extremely significantly higher than 1,2 and 3 embryonic ages (P<0.01).The expression of MSX2 gene in heart of 12 embryonic age were extremely significantly lower than that of 9 embryonic age (P<0.01).However,it showed a gradually decreasing trend in liver of 9-18 embryonic ages,and the expression were the highest in liver of 9 embryonic age (P<0.05).【Conclusion】 The sequence of MSX2 gene in Langya chickens were successfully cloned.The expression patterns of MSX2 gene were different in different embryonic ages and different tissues of the same embryonic age,which provided a foundation for further exploring the structure and function of MSX2 gene in Langya chickens.

【Objective】 The aim of this study was to screen the genes related to intramuscular fat deposition and analyze the expression characteristics based on studying the transcriptome sequencing data of longissimus dorsi muscle in Mashen and Large White pigs.【Method】 The longissimus dorsi muscle samples of Mashen pigs and Large White pigs at the age of 180 days old were collected and sequenced by RNA-Seq technology to screen differentially expressed genes.Founctional enrichment analysis of the differentially expressed genes was conducted by GO function and KEGG pathway.GeneCards was used to query gene function online and further screen differentially expressed genes related to intramuscular fat deposition.Real-time quantitative PCR was used to analyze the expression characteristics of the differentially expressed genes in different tissues of the two varieties and intramuscular adipocyte differentiation process at the different stages.【Result】 There were 280 differentially expressed genes in longissimus dorsi muscle between Mashen and Large White pigs,of which 128 were significantly up-regulated and 152 were significantly down-regulated.GO function enrichment analysis identified 46 items,including 24 in biological processe,8 in molecular function,and 14 in cellular component.Meanwhile,KEGG pathway analysis recognized that the differentially expressed genes were involved in PPAR,MAPK and adipokine signaling pathways.Six genes,TRAF2,DUSP1,ACOT4,NR4A1,SLC27A6 and PLIN5,which related to lipid deposition,were obtained by GeneCards function search.Real-time quantitative PCR results showed that the expression of NR4A1,DUSP1 and PLIN5 genes in longissimus dorsi muscle between Mashen and Large White pigs were significantly or extremely significantly different (P<0.05 or P<0.01),while the expression of TRAF2,ACOT4 and SLC27A6 genes were not significantly different (P>0.05),but the expression trend was consistent with the sequencing results.The expression of NR4A1 and PLIN5 genes in abdominal subcutaneous adipose tissue of Mashen pigs were extremely significantly higher than those in dorsal subcutaneous adipose tissue (P<0.01),whiles the expression levels of NR4A1 and DUSP1 genes in dorsal subcutaneous adipose tissue of Large White pigs were extremely significantly higher than those in abdominal subcutaneous adipose tissue (P<0.01),and the expression of PLIN5 gene of Large White pigs was significantly higher than that in abdominal subcutaneous adipose tissue (P<0.05).In the dorsal subcutaneous adipose tissues,the expression of NR4A1,DUSP1 and PLIN5 genes in Large White pigs was extremely significantly higher than those in Mashen pigs (P<0.01).In the abdominal subcutaneous adipose tissues,the expression of NR4A1 and DUSP1 genes in Large White pigs were significantly higher than those in Mashen pigs (P<0.05),and the expression of PLIN5 gene was not significantly different (P>0.05).During the porcine intramuscular adipogenic differentiation,the expression of NR4A1 gene showed a down-regulated trend,and the expression of DUSP1 and PLIN5 genes showed an up-regulated trend.【Conclusion】 In this study,three candidate genes (NR4A1,DUSP1 and PLIN5) related to porcine intramuscular fat deposition were obtained,which could provide a reference for further exploring the regulatory mechanism of porcine intramuscular fat deposition.

【Objective】 This study was aimed to investigate the effect of zinc finger BED domain-containing protein 6 (ZBED6) gene knockout on gene transcription and expression in porcine kidney,and analyze the target genes and pathways of ZBED6 gene regulating porcine kidney metabolism.【Method】 Kidney tissues were collected from 8-month-old ZBED6 gene knockout Bama Xiang pigs (ZBED6 KO) and age-matched wildtype Bama Xiang pigs (WT) (n=3).The total RNA was extracted and Real-time quantitative PCR was used to analyze the expression of insulin-like growth factor 2 (IGF2) and other differentially expressed genes.Illumina Hiseq high-throughput sequencing technology was performed to evaluate the mRNA of kidney in ZBED6 Ko and WT pigs.Sus scrofa 11.1 was used as the reference genome sequence,the differentially expressed genes were analyzed and filtered by the softwares TopHat,Cufflink,Cuffmerge and Cuffdiff.Hierarchical clustering analysis and KEGG pathway enrichment of the differentially expressed genes were also performed.The reliability of differentially expressed genes in RNA-Seq results was verified by Real-time quantitative PCR.【Result】 Real-time quantitative PCR results showed that the expression of IGF2 gene mRNA in kidney of ZBED6 KO pigs was significantly higher than that of WT pigs (P<0.05).The sequencing results showed that 78 G data was obtained,and at least 87.3% of the reads were mapped to porcine genome,suggesting the quality of sequencing was high.RNA-Seq analysis showed that a total of 25 213 genes were detected in kidney of WT and ZBED6 KO pigs.Under the criteria of adjust-P<0.05 and log₂|FoldChange|>2,299 differentially expressed genes were obtained,including 103 up-regulated genes and 199 down-regulated genes.Heatmap and PCA results showed that the gene expression pattern was similar within groups,and was different between groups.KEGG pathway enrichment analysis displayed that the differentially expressed genes were mainly involved in retinol and other metabolic pathways,especially,9 differentially expressed genes were enriched to the retinol metabolism pathway,including CYP2C42,AOX1,ENSSSCG00000036274,RDH16,CYP2A19,ENSSSCG00000022724,CYP26B1,CYP1A1 and RDH5,7 genes (CYP2C42,AOX1,RDH16,CYP2A19,CYP26B1,CYP1A1 and RDH5) of which were further validated by Real-time quantitative RCR,confirmed the reliability of RNA-Seq results.【Conclusion】 The effect of ZBED6 gene knockout on kidney metabolism of Bama Xiang pigs was analyzed by RNA-Seq technology in this study,it affected its metabolic related signal pathway by regulating the expression of multiple downstream genes in kidney,which provided materials for clarifying the function of ZBED6 gene.

【Objective】 The purpose of this study was to establish a systematic porcine leukocyte antigen-1 (SLA-1) epitope screening system using porcine renal epithelial cells 15(PK15).【Method】 Total RNA was extracted from PK15 cells,specific primers were designed,and SLA-1 gene (SLA-1*PK15) was amplified by RT-PCR method.The SLA-1 gene was further cloned into pMD18-T vector and identified by double enzyme digestion and sequencing.Bioinformatics softwares DNAMAN 5.2.2,Mega 5.0,Multalin and homology modeling were used to analyze the phylogenetic tree,secondary structure and tertiary structure.【Result】 The results showed that about 1 400 bp band was obtained by RT-PCR amplification,the results of plasmid extraction and enzyme digestion showed that SLA-1 was successfully inserted into pMD18-T vector.Sequencing results showed that the gene had a total of 1 419 bp,of which 2－1 087 bp was the coding region,encoding 361 amino acids,and the signal peptide contained 21 amino acids,which was accorded with the characteristics of SLA-1 gene.The results of evolutionary tree analysis showed that SLA-1*PK15 had the closest evolutionary relationship with SLA-1*wxd (Chinese Meishan pig) and SLA-1*0401 (Chinese Bama miniature pig),but far from SLA-1*lr02 (Danish Landrace) and SLA-1*0509 (Chinese Tibetan wild boar).The comparative analysis of extracellular amino acids showed that the main variation sites in the extracellular region of SLA-1 gene in PK15 cells and other SLA-1 genes existed in α1 and α2 regions,there were few variation sites in α3 region,and there were no characteristic amino acid variation sites.The secondary structure of SLA-1 protein of PK15 cells was mainly based on α-helix and β-fold.Homology modeling showed that SLA-1 protein of PK15 cells had a typical three-level structure of SLA class Ⅰ,α1 and α2 regions constituted the antigen polypeptide binding region.【Conclusion】 SLA-1 gene existed stably in PK15 cells.PK15 cells had potential application value as SLA-1 antigen epitope screening system.

【Objective】 The aim of this experiment was to clone the estrogen receptor α (ERα) gene sequence in Cele Black sheep,and bioinformatics analysis was carried out.At the same time,the expression of ERα gene in ovary of Cele Black sheep were measured.【Method】 The ERα gene sequence of Cele Black sheep was obtained by RT-PCR and TA cloning,and bioinformatics analysis was carried out by online softwares.The expression of ERα gene in ovary of Cele Black sheep at follicular phase,luteal phase and 30 days of pregnancy were detected by Real-time quantitative PCR.【Result】 The length of ERα gene sequence in Cele Black sheep was 1 791 bp,and encoded 596 amino acids,with the highest similarity with Capra hircus and Bos taurus.Bioinformatics analysis results showed that the molecular formula of the protein was C₂₉₃₀H₄₆₀₀N₈₂₂O₈₆₂S₄₁,the theoretical isoelectric point (pI) was 7.32,the instability index was 50.33,which was an unstable hydrophilic protein.There were 28 O-glycosylation sites and 54 phosphorylation sites,but no N-glycosylation sites.ERα protein was mainly located in nucleus and there was no transmembrane structure.The secondary structure of ERα protein was mainly alpha helix and random coil.Real-time quantitative PCR showed that ERα gene in Cele Black sheep was expressed in ovary at follicular phase,luteal phase and 30 days of pregnancy,compared with follicular phase,the expression of ERα gene in Cele Black sheep was decreased extremely significantly at luteal phase (P<0.01),and increased extremely significantly at 30 days of pregnancy (P<0.01).【Conclusion】 The length of ERα gene sequence in Cele Black sheep was 1 791 bp,and encoded 596 amino acids,which was a hydrophilic protein.The secondary structure of ERα protein was mainly alpha helix and random coil.ERα gene in Cele Black sheep was expressed in ovaries at follicular phase,luteal phase and 30 days of pregnancy.The results provided a basis for further exploration of ERα gene which played a role in ovarian follicle development,corpus luteum formation and pregnancy of Cele Black sheep.

As the most widely used antibody display technology,phage display technology has gradually become an important tool for the production of genetically engineered antibodies.Phage display technology is a molecular biology technology that uses phage or phagemid as a carrier to integrate exogenous polypeptide genes into phage genes and display the exogenous protein on the surface of the phage in the form of fusion expression.In recent years,phage display technology has been used more and more widely in the field of antibody screening.Compared with traditional methods for preparing antibodies,phage display technology has the characteristics of high throughput,low cost,and simple operation.And the antibodies screened by this technology can not only be selected and purified with the assistance of the tag protein,but also the complete gene sequence of the single-chain antibody can be obtained by the method of gene sequencing.This review first classified the phage antibody library.According to the source of the antibody,the phage antibody library could be divided into natural antibody library and immune antibody library.The specificity and positive rate of antibody libraries prepared from immunized animals were significantly higher than those of natural antibody libraries.Then the construction process of the phage antibody library was briefly described.The selection of the phage expression vector was the key to the display technology.The helper phage integrated with the phagemid gene infected its specific strains,so that the system could be selectively screened through antibiotic plates.Finally,the research progress of the application of phage display technology in the field of disease prevention and control was discussed.The first humanization of antibodies had made it possible for homologous antibodies to be used in clinical applications.Subsequent research and development of animal homologous antibodies for the prevention and treatment of viral diseases had proved the ability of phage display technology to produce diagnostic or potential therapeutic reagents.This review mainly focused on the construction of phage display system and its application in the field of disease prevention and control,in order to guide the subsequent screening of single-chain antibodies through this technology.

【Objective】 This study was aimed to investigate the variation of crude protein and mineral element concentrations of pigeon milk in squabs from 1 to 28 days.【Method】 A total of 36 pairs of parental pigeons were selected,each pair of parental pigeons fed 2 squabs.Pigeon milk samples were collected from squabs on 1,3,5,7,14 and 28 days after hatching,as soon as they were fed by the parental pigeon,the pigeon milk was forced out from the squab crop to mouth immediately.Each treatment had 6 repetitions and 2 squabs per replicate.Squabs were weighed on 1,7,14,21 and 28 days,and the feed intake of breeding pigeons was recorded weekly.【Result】 The body weight of squabs increased linearly and quadratically with age (P<0.05),the body weight of squabs increased within 1-21 d (P<0.05),and then kept stable.The pellet feed and grain intake,and the total feed intake of breeding pigeons increased significantly linearly and quadratically with age (P<0.05),they both increased significantly after 7 d (P<0.05),and remained stable within 15-28 d.The crude protein and mineral element concentrations of pigeon milk had significant differences with age (P<0.05).The content of crude protein decreased linearly and quadratically with age (P<0.05),the crude content of protein maintained stability within 1-5 d,decreased in 7 day (P<0.05),and kept stable in 14-28 d.The content of Ca increased linearly (P<0.05),while the content of P decreased linearly (P<0.05),and Ca/P increased linearly (P<0.05) with age.The contents of Cu,Mn and Zn increased linearly and quadratically with age (P<0.05),and Fe content increased quadratically with age (P<0.05).The contents of Cu,Fe,Mn and Zn maintained stability within 1-3 d.The content of Cu increased at 5 d (P<0.05),then kept stable, and increased significantly at 14 d (P<0.05),and decreased significantly at 28 d (P<0.05).The contents of Fe,Mn and Zn increased at 5 d (P<0.05),and remained unchanged in 5-28 d.However,there was no significant difference in the content of Se (P>0.05).【Conclusion】 The body weight of squabs increased within 1-21 d,and remained stable within 21-28 d.The pellet feed and grain intake,and the total feed intake of breeding pigeons increased with age.The content of crude protein and P in pigeon milk decreased with age,while the contents of Ca,Cu,Fe,Mn and Zn increased with age.

【Objective】 On the basis of adding 2% soybean oil,this experiment was conducted to investigate the effects of concentrate to forage ratio on ruminal fermentation and conjugated linoleic acid production of sheep in vitro.【Method】 Rumen fluid from sheep was used as the inoculum,and 7 levels of dietary concentrate to forage ratio (concentrate to forage ratio was 2∶8,3∶7,4∶6,5∶5,6∶4,7∶3,8∶2 respectively) were used as the substrate,2% soybean oil was added into glass fermentation tubes during the in vitro 48 h fermentation.The trail was repeated twice with four parallels in each group.【Result】 ①The concentration of acetic acid in level 4∶6 was the highest and significantly higher than those with others (P<0.01).The concentration of valeric acid increased firstly and then decreased with the increase of concentrate to forage ratio,and the concentration was hightest in level 4∶6.The concentration of total volatile acid in level 4∶6 was significantly higher than that in levels 2∶8,3∶7,6∶4 and 7∶3 (P<0.05).There was no significant difference in the concentration of propionic acid,butyric acid,isobutyric acid,isovaleric acid and the ratio of acetic acid to propionic acid between other treatment levels (P>0.05).pH value was highest at level 8∶2.②The dry matter degradation rate of level 4∶6 was significantly higher than levels 3∶7 and 5∶5 (P<0.05).The production of methane in level 4∶6 was the lowest and significantly lower than that in level 6∶4 (P<0.01).Gas production of level 6∶4 was higher than levels 2∶8,3∶7 and 5∶5 (P<0.05),but there was no significant difference with level 4∶6 (P>0.05).③The concentration of ammonia nitrogen increased first and then decreased,which the highest point located in level 4∶6,extremely significantly higher than that in levels 2∶8 and 8∶2 (P<0.01),significantly higher than that in levels 3∶7 and 6∶4 (P<0.05).The concentrations of microbial protein and acetic acid in level 4∶6 were the highest and significantly higher than those with others (P<0.01).④There was no significant difference in the concentration of conjugated linoleic acid in each group (P>0.05).【Conclusion】 On the basis of supplemented 2% soybean oil,the level of 4∶6 of dietary concentrate to forage ratio was the best value for ruminal fermentation and dry matter degradation of sheep in vitro.

【Objective】 The aim of this study was to investigate the effects of different forage grade index (GI) and carbohydrate balance index (CBI) combined diets on rumen and rectal bacterial flora of yaks,and to provide theoretical reference value for the regulation of nutrient digestion and absorption and rational dietary allocation of yaks.【Method】 Twelve healthy 3.5-year-old male yaks with similar weight were divided into three groups equally,which were corn silage-alfalfa hay-concentrate group (CAC),corn silage-wheat straw-concentrate group (CWC),and corn silage-oat hay-concentrate group (COC).All three groups of experimental yaks were fed with total mixed diet for 28 days.At the end of the experiment,rumen fluid and rectal stool samples were collected,and 16S rRNA sequencing technology was used to analyze the changes of rumen and rectum bacterial flora.【Result】 For rumen,①The Chao1 index,Ace index,Simpson index and Shannon index of the rumen bacterial flora in the three groups were not significantly different (P>0.05).②PCoA analysis showed that there was significant PC1 difference in rumen flora between CAC and COC groups (P<0.05).③At the phylum level,the Actinobacteria in rumen of CWC group were significantly different from those in CAC and COC groups (P<0.05).At the genus level,there was no significant difference in rumen bacterial flora among the three groups (P>0.05).④Rumen bacterial gene function prediction showed that:The secondary differential KEGG metabolic pathways including replication and repair,cellular community-prokaryotes,cellular processes and signaling and environmental adaptation were significantly different in the rumen bacterial flora between CAC and COC groups (P<0.05),replication and repair,transcription,nucleotide metabolism,lipid metabolism,cellular community-prokaryotes and aging were significantly different between CAC and CWC groups (P<0.05),and lipid metabolism,metabolism of other amino acid and aging were significantly different between CWC and COC groups (P<0.05).For rectum,①The Chao1 index,Ace index,Simpson index and Shannon index of the rectal bacterial flora in three groups were not significantly different (P>0.05).②PCoA analysis showed that there was no significant difference in rectal bacterial flora among three groups (P>0.05).③There was no significant difference in rectal bacterial flora among three groups at phylum and genus levels (P>0.05).④Rectum bacterial gene function prediction showed that:Energy metabolism pathway was significantly different in the rectal bacterial flora between CAC and COC groups (P<0.05),membrane transport,energy metabolism,glycan biosynthesis and metabolism,metabolism of cofactors and vitamins,cell motility,folding,sorting and degradation,transcription and environmental adaptation were significantly different between CAC and CWC groups (P<0.05),and lipid metabolism was significantly different between CWC and COC groups (P<0.05).【Conclusion】 Different GI and CBI diet combinations changed rumen bacterial flora,but rectal bacterial flora diversity was not affected.With the decrease of GI,there was a tendency to increase the abundance of rumen bacterial community,and the higher GI,the more likely it was to cause fat accumulation.

【Objective】 The experiment was conducted to investigate the effects of dietary crude protein level on growth performance,carcass traits and serum biochemical indices of male Silky fowl during 26 to 45 days of age,to investigate the crude protein requirment of Silky fowl at this stage.【Method】 1 625 26-day-old male Silky fowl were assigned to 5 groups with 5 replicates per group and 65 chickens per replicate,dietary crude protein levels were 18.5％,19.5％,20.5％,21.5％ and 22.5％,respectively.The experiment lasted for 20 days.Two chickens with close to the average body weight were selected from each replicate for slaughter on the day of the end of this experiment,serum and carcasses were collected for determining carcass traits and serum biochemical parameters.【Result】 ①Dietary crude protein level extremely significantly affected final body,average daily gain and F/G (P<0.01),and with crude protein level increasing,final body weight and average daily gain were increased linearly and quadratically (P<0.01),F/G was decreased linearly and quadratically (P<0.01).Dietary crude protein level had no significant effect on average daily feed intake and survival rate (P>0.05).②Dietary crude protein level significantly or extremely significantly affected carcass weight (P<0.01),half-eviscerated weight (P<0.01) and eviscerated weight (P<0.05),but had no significant effect on breast muscle rate,leg muscle rate and abdominal fat rate (P>0.05).With dietary crude protein level increasing,carcass weight and half-eviscerated weight were decreased linearly and quadratically (P<0.01 or P<0.05).③Dietary crude protein level extremely significantly affected the serum contents of IGF-1 and TC (P<0.01),and with dietary crude protein level increasing,the TC content was decreased linearly (P<0.05).Dietary crude protein level had no significant effects on contents of BUN and TG (P>0.05).【Conclusion】 Dietary 21.5％ crude protein level could improve growth performance,19.5％ to 20.5％ crude protein level could improve carcass traits and 18.5％crude protein level could regulate lipid metabolism.According to the estimation of quadratic curve model,it was suggested that dietary 21.41％ to 21.83％ crude protein could achieve the best growth performance at this stage.

【Objective】 The purpose of this experiment was to study the effects of formononetin on the performance,blood gas and antioxidant indexes of Yili horses in 1 000 m race,so as to provide references for the research and development of effective nutritional supplements in the training process of Yili horses.【Method】 Twelve male Yili horses with average body weight (411 kg±29 kg),average age (2.0±0.50 years old),similar performance (135.24 s±15.75 s) in the 1 000 m race and good health were selected as the research subjects and randomly divided into control group and experimental group,with six horses in each group.Under the same conditions of feeding management,dietary nutrition level and exercise training,each horse in the experimental group was fed with formononetin 8 g per day for a supplementary feeding trial of 30 days.A 1 000 m race was held on the 30th day of the test,race results were recorded and blood samples were collected via a jugular vein immediately after the race,the electrolyte level,acid-base balance and blood gas indexes were measured by blood gas analyzer,and the antioxidant indexes were measured by kit.【Result】 The supplementary feeding of formononetin could significantly shorten the race time of the 1 000 m race of Yili horse (P<0.05).There was no significant effect on plasma electrolyte level,acid-base balance and blood gas-related indicators (P>0.05).In the aspect of plasma antioxidant indexes,the supplemental formononetin could significantly increase the superoxide dismutase,catalase and total antioxidant capacity in the plasma of horses after the race,which were increased by 11.12% (P<0.05),8.45% (P<0.05) and 32.70% (P<0.05),respectively as compared with those in control group.The contents of glutathione peroxidase and malondialdehyde in the experimental group were decreased by 1.56% and 11.32% respectively as compared with those in control group,but there was no significant difference (P>0.05).【Conclusion】 Under the conditions of this experiment,the supplementary feeding of formononetin to Yili horse could significantly improve the performance of the 1 000 m race,enhance the antioxidant capacity.

As the first immune defense barrier of body,innate immune system plays a key role in the process of resisting pathogen infection.Maximize inducing the body’s protective mechanism to reduce the inflammatory damage caused by infection through stimulating the natural immune defense function of livestock and poultry by drugs or feed additives,has become one of the sustainable solutions to cope with the rampant pathogenic microorganisms and anti-microbial resistance in the current breeding environment.Being a class of biological macromolecules,Astragalus polysaccharides (APS) is one of the most immunologically active substance,and capable of various biological effects such as immune regulation,anti-virus,anti-stress,anti-oxidation,anti-tumor,etc.In clinical application,it can effectively improve the natural immune defense function of young animals and enhance the protective efficacy and growth performance of various vaccines.Therefore,the study of its regulation effect on the natural immune system of the body and its mechanism has become a research hotspot in recent years.However,due to the influence of origin,extraction and purification methods and complex chemical structure,the further research and development of APS activity are limited to some extent.The author reviewed the progress on the extraction technology and monosaccharide components of APS,and focused on the research progress of its two-way regulation for macrophages which is the main effect or cell of innate immunity,including polarization,function,inflammatory factors expression and the research progress on the intervention of macrophage-related Toll-like receptors (TLRs) signaling pathways,and pointed out the deficiencies in current research,aiming to provide references for the in-depth study of APS immunomodulatory mechanisms and the scientific basis on the promotion and application of APS in livestock and poultry breeding.

【Objective】 In this experiment,the strains isolated from the rumen juice were studied to provide basic data for the preparation of probiotic preparations.【Method】 The rumen juice of 10 healthy Holstein dairy cattle was collected,and a single strain was obtained by coating,characteristic culture and purification.The bacterial DNA was extracted and amplified by 16S rDNA PCR.After 16S rDNA gene sequencing and identification,sequence alignment was carried out and evolutionary tree was constructed to determine the species of strains.Different kinds of bacteria were cultured for 0-48 hours to determine their growth characteristics,and acid,alkali and bile salt tolerance experiments were carried out to determine their different tolerance.【Result】 20 strains were obtained by coating the isolated strains.After PCR amplification and sequencing,it was found that 20 strains belonged to 7 different bacteria.It was roughly determined that L7 was Bacillus amyloliquefaciens,K7 was Bacillus subtilis,S7 was Stenotrophomonas maltophilia,C2 was Bacillus licheniformis,M7 was Streptococcus equi,F7 was Citrobacter freundii,and T7 was Kurt gigasii.From the growth curve,it could be seen that the optimum growth time of the seven strains reached the fastest growth period in 24-36 h respectively.M7 and K7 had strong tolerance to acid,while S7,C2 and F7 were more sensitive to acid,C2 had the strongest tolerance to alkali,T7 had the strongest tolerance to bile salt,and S7 had the least tolerance to bile salt.【Conclusion】 The seven strains isolated from rumen in this study had different degrees of tolerance to acid-base and bile salt,and the optimal growth cycle.

【Objective】 This study was aimed to explore the influence of genetic variation of insulin-like growth factor 1 receptor (IGF1R) gene on the growth traits of sheep (Ovis aries),in order to provide effective molecular genetic markers for the breeding of new breeds (lines) of high-quality mutton sheep.【Method】 Using liquid-phase capture sequencing technology to obtain IGF1R gene mutation sites in a total of 91 Doper sheep,Small-tailed Han sheep and Tan sheep,and screen out single nucleotide polymorphism (SNP) with significant differences among breeds.For the selected sites,flight mass spectrometry was used to detect 383 sheep of the hybrid offspring of 3 sheep breeds,and the correlation analysis with growth traits of newborn and 3 months old was carried out.【Result】 A total of 347 mutation sites were detected in IGF1R gene of 3 sheep breeds,there were 8 SNPs in exon:g.7628315 T>C,g.7628528 T>C,g.7628690 C>T,g.7833817 C>T,g.7836687 C>T,g.7840691 T>C,g.7855904 C>G and g.7872277 C>T.which showed polymorphism in the population.χ² test results showed that all SNPs were in Hardy-Weinberg equilibrium (expect for g.7628690 C>T in Small-tail Han sheep).g.7628528 T>C and g.7872277 C>T showed low polymorphism in 3 sheep breeds (PIC<0.25);g.7833817 C>T showed moderate polymorphism in 3 sheep breeds (0.25<PIC<0.50);The other 5 SNPs showed moderate polymorphism in 1 or 2 populations.The single-site association analysis showed that g.7628528 T>C was significantly correlated with the 3 months old body weight and chest girth of F2 generation,and the newborn chest girth of H2 generation (P<0.05);g.7628690 C>T was significantly correlated with the newborn chest girth of H2 generation (P<0.05);g.7833817 C>T was significantly correlated with the 3 months old body height of F2 generation (P<0.05);g.7836687 C>T was significantly correlated with the 3 months old body weight and chest girth of F1 generation,and the 3 months old body height of F2 generation (P<0.05);g.7755904 C>G was significantly correlated with the 3 months old chest girth of F1 generation,and the newborn chest girth of F2 generation (P<0.05);g.7872277 C>T was significantly correlated with the 3 months old chest girth of H1 generation,the newborn body weight,body height,chest girth,and 3 months old body height of H2 generation (P<0.05);g.7628315 T>C was significantly correlated with the 3 months old body height and body length of F1 generation,and the 3 months old body height of H2 generation (P<0.05);g.7840691 T>C had no significant association with growth traits (P>0.05).Linkage disequilibrium analysis showed that g.7628315 T>C-g.7628528 T>C and g.7833817 C>T-g.7840691 T>C formed 2 strong linkages,3 haplotypes were constructed,and 6 genotypes were formed after combination,the dominant genotypes were H2H2 and H5H6,respectively.The association analysis of haplotype combination showed that the newborn body length of H3H1 genotype individuals was significantly higher than that of H3H2 genotype (P<0.05),and the 3 months old body height of H2H2 genotype individuals was significantly higher than that of H1H2 genotype (P<0.05),the 3 months old body length of H4H5 genotype individuals was significantly higher than that of H4H6 genotype (P<0.05),and there were no significant differences in other growth indicators among the combined genotypes (P>0.05).【Conclusion】 The results of this study indicated that the genetic variation of IGF1R gene had an significant impact on the growth traits of sheep.8 SNPs caould be considered as potential candidate genetic markers during the growth and development of sheep,which could provide information for the selection and breeding of IGF1R gene in mutton sheep,and provide the theoretical basis for the molecular marker-assisted breeding of mutton sheep.

【Objective】 This study was aimed to analyze the genetic evolutionary relationship between Xinjiang Black bee and 4 introduced Apis mellifera species,and explore candidate genes related to important germplasm characteristics of Xinjiang Black bee by detecting genome selection signal.【Method】 For Xinjiang Black bee,Apis mellifera ligustica,Apis mellifera caucasica,Apis mellifera carnica,Apis mellifera mellifera,a total of 50 Apis mellifera queen individuals from 5 species and 1 Apis cerana cerana queen were analyzed by whole genome resequencing to identify single nucleotide polymorphism (SNP) markers of Xinjiang Black bee.Apis cerana cerana was taken as reference group,the evolutionary relationship of different species was clarified by constructing Neighbor-Joining tree.SNP information was used to analyze principal component and population genetic structure and linkage disequilibrium of Xinjiang Black bee.Fst and θπ were used to detect the genome-wide selection signal between Xinjiang Black bee and other Apis mellifera populations,and the GO function and KEGG pathway enrichment of candidate genes were analyzed.【Result】 A total of 1 728 216 SNPs were identified in Xinjiang Black bee,18 526 non-synonymous mutation loci might be candidate SNPs for further study on genetic characteristics of Xinjiang Black bee;The result of phylogenetic tree and principal component analysis showed that there were some differences between Xinjiang Black bee and other species,which closely related to Apis mellifera ligustica,Apis mellifera carnica,Apis mellifera mellifera,but far related to Apis mellifera caucasica.Population genetic structure analysis showed that when K>3,Xinjiang Black bee came from an independent ancestral subpopulation,which was completely different from Apis mellifera ligustica,Apis mellifera caucasica,Apis mellifera carnica,Apis mellifera mellifera.Linkage disequilibrium analysis showed that Xinjiang Black bee groups were subjected to stronger selection intensity.The results of selection signal analysis showed that the selected genes of Xinjiang Black bee were mainly enriched in biological process related to substance metabolism,reproduction and endocytosis signaling,the cell component was enriched in the orbumin,nuclear body,chromatin and film,the molecular function was mainly enriched in organic cyclic compound binding,ion bonding and others.12 candidate genes Cyp314A1,Trichohyalin-like,CCDC112,Sorbitol dehydrogenase,SPI-3,Fer3HCH,TNS1,Pten,ADSL,Octβ2R,AFFG1 and ARHGEF17 were screened according to these molecular pathways.KEGG pathway enrichment analysis showed that the selected genes of Xinjiang black bee were mainly enriched in purine metabolism,entosis and phosphoinositol metabolism.【Conclusion】 The genomic SNP revealed the genetic structure and independent genetic background of Xinjiang Black bee,this study indicated that genes associated with cold resistance and reproduction and growth development and disease resistance in the genome of Xinjiang Black bee were subjected to strong selection pressure during variety formation.These selection signals could provide important genetics information for the genetic evolution,evaluation of Xinjiang Black bee,the protection and utilization of the creation of bee resources in China.

【Objective】 The purpose of this study was to analyze the expression profile of long non-coding RNA (lncRNA) in tail fat tissues of different tail fat-type sheep,and explore the relationship between lncRNA and tail fat deposition mechanism in sheep,so as to provide a theoretical basis for revealing the mechanism of tail fat deposition in sheep.【Method】 Xinjiang local sheep Bashibai sheep(fat tail-type) and the hybrid second generation of wild Argali×Bashibai sheep (small tail-type) were selected as the research objects.The tail fat tissues of two populations were collected,the transcriptome sequencing technology was used to screen the differentially expressed lncRNA and predict target gene,and GO function and KEGG pathway enrichment were analyzed.The expression results of transcriptome sequencing were verified by Real-time quantitative PCR.【Result】 728 differentially expressed lncRNAs were screened by differential expression analysis,of which 270 lncRNAs were down-regulated,and the expression of 458 lncRNAs was up-regulated.The target gene was screened by GO function and KEGG pathway enrichment analysis of differentially expressed lncRNA,there were 634 target genes involved in diseases,immunity,protein modification,cell metabolism and other related functions,involving a total of 76 signal pathways.Some lncRNA-mediated target genes SCD,GPAM,THRSP,FASN,etc,were related to tail fat deposition in sheep.The result of Real-time quantitative PCR and transcriptome sequencing was consistent.【Conclusion】 lncRNA played an important role in regulating tail fat deposition in the evolution of tail fat in sheep,which provided theoretical basis for analyzing fat deposition in sheep from the perspective of lncRNA.

Birds in the natural world show abundant feather colors,which plays an important role in avoiding natural enemies,predation,courtship,and resisting ultraviolet rays.Research on the color traits of chicken feathers helps to strengthen the differentiation and identification of breeds.It is particularly important to establish distinctive marks between breeds and ensure the appearance of the same breed in breeding work.Feather color is an important part of genetic research on poultry phenotype,mainly determined by melanin and carotenoids.At present,most researches on the genetic regulation mechanism of chicken feather color traits focus on melanin-related pathways.Studies have shown that Wnt,KIT/KITL and EDN3/EDNRB signaling pathways have important regulatory effects on the growth,migration and differentiation of melanomas,and α-MSH/ASIP-MC1R signaling pathway is responsible for regulating the synthesis of melanin.Existing studies have shown that it can reveal the genetic regulation mechanism of various feather traits such as black feather,stippled feather,dominant white feather,recessive white feather,autosomal albinism,silver feather,sex-linked imperfect albinism,dark brown feather,lavender feather (grey feather),tyrosinase-independent recessive white feather,mottled feather and sex-linked barring feather (Lu Hua feather) by genome-wide genetic variation detection technology,mining genes and variation loci related to feather color traits.Additionally,the Turing pattern was used as a theoretical basis to explain the formation mechanism of complex feather patterns.By summarizing the genetic regulation mechanism of melanin-related regulatory pathways and Turing pattern on chicken’s feather color traits,the authors reviewed the related research on chicken’s feather color traits that have identified loci in recent year to provide insights into the research on the molecular mechanism of chicken’s feather color and the marker-assisted selection of chicken’s feather color traits in the breeding process.

Non-coding RNA (ncRNA) refers to RNA transcribed from the genome,which does not encode proteins,but can perform certain biological functions at the RNA level.There are many kinds of non-coding RNA,and different kinds lead to different functions.Hair follicle is a special appendage of cashmere goat skin,which is located in the dermis of the skin and occurs in the embryonic stage of cashmere goat.Due to the transmission of signal molecules between dermal cells and epithelial cells,its development changes periodically,going through growth stage,degenerative stage and resting stage.In recent years,there are many reports on the role of ncRNA in the development of cashmere goat hair follicle.This paper summarizes the biogenesis of microRNA (miRNA),long-chain non-coding RNA (lncRNA) and circular RNA (circRNA) and their mechanisms in the development of cashmere goat hair follicles.miRNA is a ncRNA molecule with a length of 18-25 nt in eukaryotes,it can inhibit post transcriptional gene expression by specifically binding to target mRNA.In the development of cashmere goat hair follicles,miRNA regulates the cycle and regeneration ability of hair follicles by inhibiting the genes related to hair follicle development and the key molecules of related signal pathways.lncRNA is a ncRNA with a length of more than 200 nt,after splicing,it has a polyA tail and promoter structure,which can promote the proliferation and differentiation of hair follicle cells,and regulates the cycle activity of hair follicle development and villus growth by regulating the targeted expression of genes and interacting with a variety of signal pathways.circRNA is different from the traditional linear RNA,which is a closed circular RNA and contains many miRNA binding sites.It plays the role of miRNA molecular sponge in cashmere goat hair follicle development and villus growth,that is,through the mediation of miRNA,it regulates the expression of target genes,promotes the differentiation of hair follicle stem cells into secondary hair follicles,and then relieves the inhibitory effect of miRNA related to hair follicle development and villus growth on its target genes,to improve the expression level of target genes.Through the research review of miRNA,lncRNA and circRNA,it is hoped to provide a reference for the construction of the molecular regulation network of hair follicle growth and the in-depth study on the regulation mechanism of the hair follicle development,and provide a theoretical basis for the better use of modern molecular biology technology to improve the quality of cashmere in the future.

【Objective】 This study was aimed to screen the antagonistic bacteria of Aspergillus flavus,and explore its optimal culture conditions and effective antibacterial components,so as to provide a new theoretical basis for the application of biological control methods to inhibit Aspergillus flavus pollution in practical production.【Method】 The strains to be tested were screened and identified by plate confrontation method and molecular biology identification method.Taking the inhibition rate as the measurement standard,the optimal culture temperature,culture time,inoculation amount,rotating speed,pH and liquid loading of Aspergillus flavus antagonistic bacteria were determined.In addition,the protein was extracted from the sterile fermentation supernatant of antagonistic bacteria by ammonium sulfate precipitation method,and the effect of the crude extract on the growth of Aspergillus flavus was observed.Then,the crude protein was further purified by ion exchange chromatography and gel filtration chromatography,and mass spectrometry sequencing and sequence comparative analysis were performed to screen and identify the effective antibacterial components produced by antagonistic bacteria.【Result】 A strain of Bacillus amyloliquefaciens B10 was screened (strain preservation No.CCTCCNO:M2018353).The optimum culture conditions were as follow:Temperature was 40 ℃,culture time was 36 h,inoculation amount was 3%,rotating speed was 120 r/min,pH was 6.0,liquid volume was 30 mL,and the maximum inhibition rate was 46.56%.In addition,the crude protein could effectively inhibit the growth of Aspergillus flavus and destroy the normal morphology of mycelium.Eight possible effective antibacterial components such as chitin binding protein,chitosanase and glucanase had been selected.【Conclusion】 A strain of Bacillus amyloliquefaciens B10,which inhibited the normal growth and development of Aspergillus flavus,was isolated and identified and its various protein components had significant antibacterial activity against Aspergillus flavus.

【Objective】 The aim of this study was to investigate the anti-apoptotic effect of canine adipose-derived mesenchymal stem cells (cAd-MSCs) on in vitro model of severe acute pancreatitis (SAP),in order to provide a theoretical guide for the treatment of pancreatitis with stem cells.【Method】 ①Type Ⅰ collagenase digestion method was used to separate cAd-MSCs,the expression of stem cell markers CD29,CD34,CD44,CD45,CD73 and CD90 was identified by flow cytometry,and its multidirectional differentiation potential was identified by adipogenic,osteogenic and chondrogenic differentiation.②Pancreatic acinar cells (PACs) were isolated from mouse pancreatic tissue with type Ⅰ collagenase.The expression of pancreatic duct-specific gene CK19,β-islet cell-specific gene Insulin-1,α-islet cell-specific gene Glucagon and PAC-specific genes PTF-1α,CPA-1 and AMY2B in PACs and pancreatic tissues were detected by Real-time quantitative PCR.③PACs were treated with 10 and 20 μg/mL lipopolysaccharide (LPS),10 and 100 mmol/L Caerulein,and 10 μg/mL LPS+100 mmol/L Caerulein,the cells cultured without drugs were used as the control group.The survival rate of PACs was detected by CCK-8 at 24 h to screen the optimal treatment group for constructing endoplasmic reticulum(ER) model in vitro (model group,P).The survival rate of control group (Naive) and P group were detected by CCK-8 at 0,2,4,8,12 and 24 h,the expression of ER stress-related proteins in P group were detected by Western blotting.④To determine the mode of action of cAd-MSCs on PACs,the experiment was divided into PAC group (only PACs,Naive),P group,indirect co-culture group (IC) and direct co-culture group (DC).The expression of TNF-α gene was detected by Real-time quantitative PCR.⑤In the IC system,cells were divided into blank control group (PACs only,Naive),control group (PACs co-cultured with cAd-MSCs),P group and experimental group (drug-treated PACs co-cultured with cAd-MSCs,T).The expression of ER stress-related genes and proteins were detected by Real-time quantitative PCR and Western blotting at 12 h.The apoptosis of cells in each group was detected by TUNEL assay.【Result】 ①The isolated and cultured cAd-MSCs showed fibroblast-like cell morphology,highly expressed stem cell markers CD29,CD44,CD73 and CD90,did not express CD34 and CD45,and had the ability of adipogenic,osteogenic and chondrogenic differentiation.②The isolated PACs showed cobblestone-like morphology,and compared with pancreatic tissue,the expression of AMY2B,CPA1,PTF-1α genes were significantly increased (P<0.05),CK19,Glucagon,and Insulin-1 were extremely significant decreased (P<0.01).③Compared with control group,the cell viability in 10 μg/mL LPS+100 mmol/L Caerulein group was extremely significant decreased (P<0.01),which was selected as the best treatment group for building an ER stress model.Compared with control group,the cell viability of PACs in P group was significantly decreased at 4 h (P<0.05),and extremely significant decreased at 8,12 and 24 h (P<0.01).Western blotting results showed that the expression of Grp78,CHOP and Caspase-12 protein were increased significantly from 4 h (P<0.01).④Compared with Naive group,the mRNA expression of TNF-α gene in P group was extremely significantly increased (P<0.01).Compared with P group,the expression of the TNF-α gene in IC and DC groups was extremely significantly decreased (P<0.01).The subsequent experiments were carried out with IC system.⑤In the IC system,compared with P group,the relative mRNA and protein expressions of Grp78,Caspase-12 and CHOP in T group were extremely significantly decreased (P<0.01).The TUNEL results showed that the number of positive cells in T group was obviously reduced.【Conclusion】 In this experiment,ER stress model of SAP in vitro were sucessfully constructed,and it was confirmed that cAd-MSCs could protect the ER stress of PACs.

【Objective】 It was aimed to isolate and screen probiotic lactic acid bacteria from the reproductive tract of healthy dairy cows,and to provide probiotic strains for the prevention and treatment of dairy cow endometritis in the future,and lay the foundation for the development of microecological preparations for the prevention and treatment of this disease.【Method】 The reproductive tract secretion samples of 10 healthy dairy cows were collected and isolated and cultured with MRS medium.The isolated strains were identified by morphological observation and 16S rDNA sequence.The sequence results were compared with the reference strains on NCBI nucleic acid database to determine the species of each isolated strain.Oxford cup method was used to isolate and screen strains with good antibacterial activity.The growth curve,acid production ability and antimicrobial sensitivity of these screened strains were studied,and their main antibacterial substances were detected.【Result】 A total of 13 strains of lactic acid bacteria were isolated from 10 samples.After staining microscopy and 16S rDNA sequencing analysis,13 strains of lactic acid bacteria were identified as 11 strains of Lactobacillus plantarum,1 strain of Enterococcus specialis and 1 strain of Enterococcus rodentis respectively.5 strains of lactic acid bacteria with good antibacterial ability against Escherichia coli were obtained by single strain antibacterial test.The growth characteristics and acid production ability of the five strains of lactic acid bacteria were good,and they were highly sensitive to ampicillin,tetracycline,erythromycin,rifampicin,trimethoprim,chloramphenicol and penicillin.The antibacterial substances of strains 21 and 35 were hydrogen peroxide and bacteriocin,while the antibacterial substances of strains 25,33 and 34 included organic acids in addition to hydrogen peroxide and bacteriocin.【Conclusion】 Three strains of lactic acid bacteria isolated from the reproductive tract of dairy cows were expected to be used as probiotics in the clinical prevention and treatment of dairy cow endometritis,and its specific prevention and treatment effect needed to be further explored.

【Objective】 This study was aimed to explore the role of transforming growth factor-β 1(TGF-β1)/Smad pathway in liver fibrosis of ascites syndrome in broilers and the prevention and treatment mechanism of compound traditional Chinese medicine water extract.【Method】 258 healthy Ross broilers were routinely raised to 7 days of age and randomly divided into 5 groups:Control group (C),model group (M,multi-factor modeling with low temperature,high-fat and high-protein feed,and high-sodium drinking water),compound traditional Chinese medicine water extract high,medium and low dose groups (T1,T2,T3,based on the model group,added 2.0,1.0 and 0.5 g/kg compound traditional Chinese medicine water extract to drinking water every day,respectively).Clinical symptoms and necropsy changes were observed,HE and Masson staining methods were used to observe the changes of liver tissue structure and collagen fiber deposition in broilers,and the weight at 15,25,35 and 45 days of age were calculated.The relative expression of TGF-β1,transforming growth factor β receptor Ⅰ(TβRⅠ),Smad2,Smad3 and Smad7 genes were detected by Real-time quantitative PCR,and the protein contents of TGF-β1 and TβRⅠ were detected by ELISA in liver at 15,25,35 and 45 days of age.【Result】 Compared with control group,the broilers in model group had enlarged abdomen and fluctuating touch pressure.There was a large amount of light yellow liquid in the abdominal cavity.The liver was congested and swollen,and the texture was brittle,with blutene chloaides on the surface.The liver tissue structure was disordered,and inflammatory cells infiltrated.A large amount of collagen fibers deposited and the liver fibrosis.The body weight in model group was extremely significantly decreased than control group (P<0.01).Compared with control group,expect for TβRⅠ protein at 15 days of age,the relative mRNA expression and protein content of TGF-β1 and TβRⅠ in liver of broilers in model group were significantly increased (P<0.01;P<0.05),the relative expression of Smad2 and Smad3 genes mRNA in liver of broilerss in model group were extremely significantly increased (P<0.01),the relative expression of Smad7 gene mRNA in liver of broilers in model group was extremely significantly decreased (P<0.01).Compared with model group,the results of the compound traditional Chinese medicine water extract groups were improved to varying degrees and high dose group had more significant effect.【Conclusion】 Liver fibrosis was involved in the occurrence and development of ascites syndrome broilers,and the mechanism was related to TGF-β1/Smad pathway.The compound traditional Chinese medicine water extract made from Astragalus,Angelica,Salvia,tuckahoe,licorice,etc.could regulate the TGF-β1/Smad pathway,inhibit the occurrence of liver fibrosis,and effectively prevent ascites syndrome.

【Objective】 The aim of this expriment was to study the effect of inlK gene on biofilm formation ability of Lm90SB2 strain and the relationship between biofilm and disinfectant resistance,in order to provide reference for effective prevention and control of Listeria monocytogenes contamination.【Method】 Listeria monocytogenes Lm90SB2 was used as the test strain.According to the inlK gene sequence (accession number:AE017262) of Listeria monocytogenes F2365 published in GenBank,specific primers for amplifying the upstream and downstream homology arm fragments of inlK gene and verifying the deletion strains were designed using Primer Premier 5.0 software.The inlK gene deletion strain was constructed by homologous recombination technology,and the deletion strain was detected by PCR with paralateral primers.The standard strain Lm90SB2 and the constructed deletion strain were cultured for 8,12,24 and 48 h and stained with crystal violet.Morphological changes were observed under an inverted microscope,and biofilm formation ability was measured with a microplate reader.PBS solution containing 3 g/L lecithin+3 g/L Tween 80 and PBS solution containing 10 g/L lecithin+20 g/L Tween 80 were used as neutralizers of bromogeramine,PBS solution containing 5 g/L sodium thiosulfate+5 g/L lecithin+20 g/L Tween 80 and PBS solution containing 10 g/L sodium thiosulfate+30 g/L lecithin+20 g/L Tween 80 were used as neutralizers of 84 disinfectant,disinfectants+bacterial suspension,disinfectant+bacterial suspension+neutralizer,neutralizer+bacterial suspension,disinfectant+neutralizer+bacterial suspension,diluent+bacterial suspension (positive control),diluents+neutralizer+culture medium (negative control) groups designed to screen neutralizers.And the sterilization rates of biofilm of two strains treated with different concentrations of bromogeramine (1∶15,1∶30) and 84 disinfectant (1∶50,1∶100) for 1,5,10 and 20 min were detected,respectively.【Result】 PCR results showed that the deletion strain Lm90SB2ΔinlK was successfully constructed,and the deletion of inlK gene resulted in significant or extremely significant decrease in the biofilm formation ability of Lm90SB2 strain (P<0.05;P<0.01).The neutralizer composed of PBS solution containing 3 g/L lecithin+3 g/L Tween 80 could effectively neutralize the bromogeramine,and PBS solution containing 5 g/L sodium thiosulfate+5 g/L lecithin+20 g/L Tween 80 could effectively neutralize 84 disinfectant.The sterilization rate of different proportions of bromogeramine (1∶15,1∶30) and 84 disinfectant (1∶50,1∶100) disinfectant on the biofilm of Lm90SB2ΔinlK strain was higher than that of Lm90SB2 strain at 1,5 and 10 min (P<0.05;P<0.01),and the sterilization rate was 100% at 20 min.【Conclusion】 The biofilm formation ability of Lm90SB2 strain and its resistance to disinfectant were decreased by deleting inlK gene.

【Objective】 This study was aimed to explore the anti-inflammatory effect of 'Ruyan Ning’ water extract,and provide theoretical basis for its clinical application and further research.【Method】 The anti-inflammatory effects of xylene-induced auricle swelling,acetic acid-induced increased peritoneal capillary permeability,carrageenan-induced foot swelling,cotton ball-induced granulation swelling,and serum cytokines contents in mice were studied.60 Kunming mice,half male and half female,were randomly divided into 5 groups,namely blank control group (normal saline group),positive control group (aspirin group),and 'Ruyan Ning’ water extract low (4 mg/g),medium (8 mg/g) and high (16 mg/g) dose groups, 12 mice in each group.Mice in each group were intragastrically administered at a fixed time every day,once a day,for 10 consecutive days.Follow-up according to the specific operation of different experiments,after the experiment,the auricle weight,D590 nm value of peritoneal washing fluid,foot weight,cotton ball granuloma weight and serum contents of tumor necrosis factor-α (TNF-α) and interleukin-1β(IL-1β) were measured.【Result】 The results of the xylene-induced auricle swelling test showed that compared with blank control group,the drug treatment group could extremely significantly reduce the auricle swelling caused by xylene (P<0.01).Compared with positive control group,there was no significant difference in auricle weight in the high-dose group (P>0.05).The results of the acetic acid-induced peritoneal capillary permeability test showed that,compared with the blank control group,the drug treatment group had a very significant inhibitory effect on the increase of acetic acid-induced peritoneal capillary permeability (P<0.01).Compared with positive control group,the capillary permeability of each dose group of 'Ruyan Ning’ water extract was significantly or extremely significantly increased (P<0.05;P<0.01).The results of the carrageenan-induced foot swelling test showed that,compared with the blank control group,the drug treatment group had extremely significant inhibitory effect on the foot swelling of mice (P<0.01).Compared with the positive control group,there were extremely significant differences in the degree of foot swelling in each dose group of the 'Ruyan Ning’ water extract (P<0.01).The results of the cotton ball-induced granulation test showed that,compared with the blank control group,the drug treatment group had a very significant inhibitory effect on the inhibition of cotton ball granulation in mice (P<0.01).Compared with the positive control group,the difference between the low and medium dose groups of the 'Ruyan Ning’ water extract was extremely significant or significant (P<0.01;P<0.05),and the high dose group of 'Ruyan Ning’ had the same effect as the positive control group (P>0.05).The results of the detection of serum cytokines in mice showed that compared with the blank control group,except for the low dose 'Ruyan Ning’ water extract group,the contents of TNF-α and IL-1β in serum in the drug treatment group were reduced to varying degrees,'Ruyan Ning’ water extract low dose group had no significant difference in the content of TNF-α in serum (P>0.05),but the content of IL-1β in serum decreased significantly (P<0.05).The content of IL-1β in serum of medium dose group and high dose group were significantly decreased (P<0.01),and the content of TNF-α in serum was significantly decreased in medium dose group (P<0.05),and extremely significantly decreased in high dose group (P<0.01).Compared with the positive control group,there was no significant difference between the two cytokines in serum of the 'Ruyan Ning’ water extract medium and high dose groups (P>0.05),and two cytokines in the serum of the low dose group had extremely significant differences (P<0.01).【Conclusion】 'Ruyan Ning’ water extract had good anti-inflammatory effect on mouse.

【Objective】 The aim of this study was to investigate the alleviating effect and mechanism of c-Jun N-terminal kinase (JNK) inhibitor SP600125 on the pharmacological damage of primary hepatocytes in piglets.【Method】 Piglet primary hepatocytes were obtained by two-step perfusion method,and the hepatocytes were divided into control group (C),JNK inhibitor group (SP),model group (M) and treatment group (T),with 6 replicates in each group.The cells in control group were not added with drugs,SP group cells were treated with 2 μmol/L SP600125,M group cells were treated with 80 μg/mL lipopolysaccharide (LPS) +20 μg/mL enrofloxacin (ENR),T group cells were treated with 80 μg/mL LPS+20 μg/ mL ENR+2 μmol/L SP600125.After treatment for 12 h,supernatants were collected to determine alanine transaminase (ALT) and glutamic oxalacetic transaminase (AST) activities,and cells were collected to determine the activities of glutathione peroxidase (GSH-Px),superoxide dismutase (SOD), and the content of malondialdehyde (MDA) and the relative expression of hepatocyte nuclear factor 1 (HNF-1) and glutathione S-transferase alpha 1 (GSTA1) mRNA.【Result】 Compared with control group,the activities of ALT and AST in hepatocyte culture medium were significantly increased in M group (P<0.05),the activities of GSH-Px,SOD and the relative expression of HNF-1,GSTA1 mRNA in hepatocytes were extremely significantly decreased in M group (P<0.01),and MDA content in hepatocytes was extremely significantly increased (P<0.01).Compared with M group,the activities of ALT and AST in hepatocyte culture medium were significantly decreased in T group (P<0.05),the activities of GSH-Px,SOD and the relative expression of HNF-1,GSTA1 mRNA in hepatocytes were significantly increased in T group (P<0.05),and MDA content was significantly decreased (P<0.05).【Conclusion】 JNK inhibitor SP600125 could alleviate the drug-induced injury of piglet primary hepatocytes caused by LPS/ENR by regulating the antioxidant capacity and the expression of HNF-1 and GSTA1 of cells.

Polysaccharide can promote immune regulation with multi-target,multi-function and multi-factor effects,so it can be widely used as an adjuvant of veterinary vaccine.As vaccine adjuvant,polysaccharide can improve the index of immune organs such as spleen,thymus and bursa of Fabricius of immunized animals,enhance phagocytic activity and antigen presentation ability of macrophages,enhance the killing ability of NK cells and promote the maturation and differentiation of dendritic cells and enhance the ability of antigen uptake,affect the type and number of T cell subsets and change the type of Th1 and Th2 immune response,promote the proliferation of B lymphocytes and stimulate the production of cytokines and antibodies.As a ligand,polysaccharide mainly binds with receptor molecules such as Toll like receptor (TLR),mannose receptor (MR),C-type lectins receptor (CLR),scavenger receptor (SR) and so on to activate downstream signal pathways and exert the above immune activities.In this paper,the mechanism of polysaccharide as an adjuvant of veterinary vaccine was discussed,in order to provide a reference for the further development and application of polysaccharide.

microRNAs (miRNAs) are a class small non-coding RNAs with short single chain.Both during normal development and the occurrence and development of disease,miRNA can bind to the target mRNA,participate in post-transcriptional regulation of genes and play an important role.Recently,with the in-depth study of miRNAs,more and more evidence showed that pathogen infection will change the expression level of host miRNAs.These differentially expressed miRNAs can actively participate in the occurrence and development of the disease,and participate in immune function,autophagy,inflammation,metabolism and other biological processes to inhibit or promote the development of the disease by inhibiting target genes expression.In addition,host miRNAs as a small RNA involved in post-transcriptional regulation,during the pathogens infection process,chicken miRNAs not only target their own genes to regulate chicken innate immune signals but also target pathogenic genes to affect pathogen adsorption,invasion,proliferation,and so on.This paper mainly introduces the biosynthesis and function of miRNAs and the regulatory role and mechanism of chicken miRNAs in the invasion and pathogenesis of some viruses,bacteria and parasites,and briefly describes the regulation strategies of chicken miRNAs after different pathogens infection.This paper reviews the biogenesis and function of miRNAs and miRNA regulatory mechanism during pathogen invasion and pathogenic process,briefly describes the regulation strategy of chicken miRNAs after infection with different pathogens,to provide some references for the diagnosis,treatment,prevention and control of chicken disease from the point of view of chicken miRNAs.

【Objective】 This experiment was conducted to investigate the optimal conditions of oxidative damage induced by H₂O₂ in goat mammary epithelial cell (GMEC),and construct a reliable and stable oxidative damage model,in order to provide model conditions for exploring the protective mechanism of anthocyanins on oxidative damage in Dioscorea alata L.【Method】 In this experiment,two factors complete random design was used,the first factor was the concentration of H₂O₂ (0 (control group),350,430,460,490,520 and 550 μmol/L),and the second factor was the treatment time of GMEC (8,11,14 and 17 h),respectively.Treatment groups with cell survival rate lower than 60% were discarded,and treatment concentration and time were readjusted according to the results.Subsequently,the activity of glutathione peroxidase (GSH-Px),the content of reactive oxygen species (ROS) and malondialdehyde (MDA) in cells,the activity of lactate dehydrogenase (LDH),catalase (CAT),superoxide dismutase (SOD) and the total antioxidant capacity (T-AOC) in culture medium were measured by the kit,so as to determine the best treatment mode of H₂O₂.【Result】 Compared with control group,the survival rate of GMEC in all treatment groups were significantly decreased after 8 h treatment (P<0.05).The survival rate of GMEC in 360,430 and 460 μmol/L treatment groups were reduced to 64.6%,72.9% and 66.2% after 8 h treatment,which basically met the screening requirement of cell survival rate (70%).Therefore,350,430,460,490 and 520 μmol/L H₂O₂ treatments were selected for 4,6,8,10 h for follow-up tests.After adjustment, the LDH activity in all treatment groups was significantly higher than that of control group at different treatment times (P<0.05), and gradually increased with the extension of time, except 430 μmol/L treatment group for 4 h. The LDH activity of all treatment groups reached the maximum value at 10 h, and was proportional to the concentration. Compared with control group, ROS content in all treatment groups at 6 h was significantly increased (P<0.05), and ROS content in all treatment groups at 8 h was significantly increased compared with 6 h (P<0.05), indicating that 8 h was the turning point of ROS content change. Compared with control group, MDA content in 430, 490 and 520 μmol/L treatment groups was significantly increased after 8 h treatment (P<0.05), and MDA content in 430 μmol/L treatment group was higher than that of control group (P<0.05). The activities of CAT, SOD, GSH-Px and T-AOC in all groups were decreased to varying degrees from 6 to 8 h after treatment, and only the activities of CAT, SOD, GSH-Px and T-AOC in 430 μmol/L treatment group were significantly lower than that of control group at 8 h (P<0.05).【Conclusion】 Therefore,the method that GMEC treated with 430 μmol/L H₂O₂ for 8 h met the need of constructing oxidative damage model.

In recent years,hypoxic cell culture has gradually become a research hotspot.It has been reported that hypoxic conditions can improve cell expansion in vitro growth kinetics,cell proliferation rate and stem cell differentiation and other functions.Hypoxia inducible factor-1α (HIF-1α) is a key factor in response to hypoxia stress and also a transduction center mediating hypoxia signal.Vascular endothelia growth factor (VEGF),hypoxia-induced mitogenic factor (HIMF) and erythropoietin (EPO) and so on are important target genes and their interactions play an important role in cell development under hypoxia conditions.In this paper,the research status of hypoxic cultured cells and the regulation mechanism and role of hypoxia-related factors were summarized,so as to provide theoretical data for exploring the mechanism of cell hypoxia,and provide more research ideas for the adaptation to altitude hypoxia and the treatment of altitude diseases.

【Objective】 This study was aimed to construct knockout mixed lineage kinase domain-like (MLKL) PK-15 cell line,and investigate the effects of MLKL gene knockout on the replication of Pseudorabies virus (PRV).【Method】 The MLKL-sgRNA editing vector was constructed by designing specific editing sites based on MLKL sequences using CRISPR/Cas9 gene editing technology,and transfected into PK-15 cell.The polyclonal cell line was obtained by puromycin screening,and PK-15 MLKL-KO monoclonal cell line was obtained by limited dilution method.The knockout level of MLKL gene on PK-15 cell was verified by target genomic PCR,sequencing and Western blotting.Viral proliferation levels were detected using Reed-Muench method.PI staining and fluorescence microscopy were used to observe cell necrosis.【Result】 The results showed that one PK-15 cell line with a 647 bp deletion of MLKL gene was screened,and the expression of MLKL protein was not detected by Western blotting.Compared with PK-15 cell,PK-15 MLKL-KO cell extremely significantly or significantly increased the viral titers of PRV GD-WH (expect for 36 h post infection) and PRV Bartha-K61 (P<0.01 or P<0.05).After PRV GD-WH and PRV Bartha-K61 were infected with PK-15 MLKL-KO cells,the necrotic cells were significantly reduced.【Conclusion】 The MLKL gene knockout PK-15 cell line was constructed in this study.Compared with PK-15 cell,PK-15 MLKL-KO cell significantly improved the replication and survival of PRV GD-WH and PRV Bartha-K61,providing a feasible strategy to increase virus yield during PRV Bartha-K61 vaccine production.

【Objective】 The purpose of the experiment was to study the regulation of long noncoding RNA (lncRNA) on autophagy in the process of Porcine epidemic diarrhea virus (PEDV) infecting African green monkey kidney cells (Vero-E6),so as to provide a new idea for the development of new drug targets against PEDV.【Method】 According to the human published lncRNA-HOTAIR gene sequence (accession No.:NR_047517.1) in GenBank,the monkey homologous sequence of lncRNA-HOTAIR (lncRNA-M) was screened by LongMan online tool,and the secondary structure and nucleocytoplasmic distribution were predicted by RNAfold and LncLocator online prediction tools.The Vero-E6 cell model infected by PEDV was established.The cells were collected at 0,6,12,24,36 and 48 h,and Real-time quantitative PCR was used to detect microtubule associated protein 1 light chain 3 (LC3),in order to determine the occurrence of autophagy at different time points after virus infected cells.The expression levels of lncRNA-M in Vero-E6 cell model infected by PEDV at 0,6,12,24,36 and 48 h were detected by Real-time quantitative PCR.Three lncRNA-M interference fragments siRNA-1,siRNA-2 and siRNA-3 were designed,synthesized and transfected into Vero-E6 cells respectively.When the confluence of Vero-E6 cells reached 90%,the cells were infected with PEDV,and after infection for 48 h,the expression of lncRNA-M was detected by Real-time quantitative PCR,and the interference fragment with the highest interference efficiency was screened.After transfecting the interference fragment with the highest interference efficiency into Vero-E6 cells,and the cells were infected with PEDV,the expression level of LC3 protein at 24 and 48 h after infection was detected by Western blotting to verify the occurrence of autophagy.【Result】 The homologous sequence of monkey lncRNA-HOTAIR was successfully screened as lncRNA 0.1,and the RNA minimum free energy secondary structure of lncRNA 0.1 was generated according to the prediction results of RNAfold.The prediction results of Lnlocator showed that the distribution of lncRNA 0.1 in nucleus and cytoplasm were 41.88% and 37.78%,respectively.48 h after PEDV infected Vero-E6 cells,the cells shrank and clustered,and the cell membrane fused to form syncytia body.1.0% agarose gel electrophoresis showed that the target band appeared at 1 326 bp,indicating that the PEDV infection Vero-E6 cell model was successfully established.In Vero-E6 cell model infected by PEDV,Western blotting results showed that the expression of LC3Ⅱ was increased in the experimental group at 6,12,24,36 and 48 h,the ratio of LC3Ⅱ /LC3Ⅰ was the highest at 48 h and was extremely significantly higher than that of control group (P<0.01).Real-time quantitative PCR results showed that the mRNA expression of LC3 was the highest at 48 h and was extremely significantly higher than that of control group (P<0.01),autophagy was activated.The expression of lncRNA 0.1 showed an upward trend,and the expression was the highest at 48 h,and the expression of lncRNA 0.1 at 48 h test group was extremely significantly higher than that in control group (P<0.01).Real-time quantitative PCR showed that siRNA-2 had the best interference effect,and the interference efficiency was 80%.After interfering with lncRNA 0.1,Western blotting results showed that the expression of LC3 protein was decreased and autophagy was inhibited in 24 and 48 h interference groups.【Conclusion】 PEDV infection could induce Vero-E6 autophagy,and lncRNA 0.1 played a role in promoting autophagy during infection.

【Objective】 The purpose of this study was to investigate the effects of Vibrio cholerae ghosts (VCG) and chitosan gel (Gel) as compound adjuvants on enhancement of immunity of inactivated elementary body (EB) against Chlamydia psittaci infection.【Method】 Sixty SPF chickens aged 7 days were randomly divided into 4 groups,15 birds per group,including EB＋VCG＋Gel group,EB＋VCG group,Gel group and EB group.SPF chickens were immunized twice via nasal drop at an interval of 14 days.Specific IgG antibody levels,lymphocyte proliferation index,proportion of CD4^＋/CD8^＋ T cells,contents of cytokines (IL-4,IL-10,IL-12 and IFN-γ) were determined post immunization.Afterwards,throat bacteria excretion and lung lesions were detected post challenge with virulent Chlamydia psittaci strain.【Result】 Compared with Gel and EB groups,IgG antibody level,lymphocyte proliferation index and IFN-γ content and the ratio of CD4^＋/CD8^＋T cells in EB＋VCG＋Gel and EB＋VCG groups were significantly or extremely significantly increased (P<0.05;P<0.01).In addition,compared with Gel and EB groups,on the 12th day after challenge,the amount of laryngeal bacteria excretion in EB＋VCG＋Gel group was extremely significantly lower (P<0.01),on the 7th day after challenge,the lung lesion was extremely significantly lower (P<0.01).【Conclusion】 The novel nasal vaccine containing chitosan gel,VCG and inactivated EB was a promising approach by enhancing humoral and cellular responses,blocking transition via respiratory tracts,contributing to eradication the transmission via infected animals to human beings.

【Objective】 The purpose of this study was to establish an indirect ELISA method based on lipoprotein P60 of Mycoplasma ovipneumoniae.【Method】 Bioinformatics softwares were used to analyze the antigenicity of P60 encoded proteins,and the region with the best antigenicity was screened out.After the target gene was amplified,the recombinant expression vector was constructed and the gene was sequenced,the correctly sequenced plasmid was transformed into Escherichia coli BL21(DE3) competent cells.The expression of the best antigenity protein was induced by IPTG and the induction time was optimized.SDS-PAGE was used to analyze the molecular weight of the protein and its expression form.Using recombinant protein as antigen and positive serum of Mycoplasma ovipneumoniae as antibody,Western blotting was used to analyze its reactivity.An indirect ELISA method was established to detect the best antigenicity protein by using the recombinant protein as the coating antigen.Twenty negative serum samples were detected by this method,and the critical value of positive and negative was calculated.The specificity,sensitivity and repeatability of this method were tested.The established indirect ELISA method and indirect hemagglutination were used to detect 92 sheep clinical serum samples.【Result】 According to DNAStar analysis,the average antigenic index of 58-928 amino acid region of P60 protein ranged from 0.8 to 1.7,and hydrophilic index ranged from 0 to 2.0,indicating that the region P60_58aa-928aa had strong antigenicity and hydrophilicity.The P60_58aa-928aa gene was amplified by PCR,and the recombinant expression vector was constructed.Gene sequencing proved that the recombinant expression vector was consistent with the expected results.SDS-PAGE results showed that when IPTG was induced at the concentration of 1 mmol/L and the induction time was 10 h,the protein expression level was higher,the molecular weight of protein was 42 ku,and it was expressed as inclusion body in Escherichia coli.Western blotting results confirmed that the recombinant protein had good reactivity.The optimal ELISA conditions showed that the concentration of antigen coating was 0.5 μg/mL,the optimal dilution concentration of primary antibody was 1∶100,the optimal incubation time was 1.5 h,and the optimal dilution ratio of enzyme-conjugate secondary antibody was 1∶4 000.The critical value of positive and negative serum was 0.36.The standard positive serum of Foot-and-mouth disease virus (FMDV),Peste des petits ruminants virus (PPRV),Brucella,Mycoplasma bovis (Mb) and Mycoplasma filamentum goat subspecies (Mmc) were all negative,which proved that the method had high specificity.In the sensitivity test,the result was still positive at the serum dilution ratio of 1∶2 048.The coefficient of variation of intra-batch and inter-batch repeated tests were all less than 10%,which proved that the method had good repeatability.The established indirect ELISA method and indirect hemagglutination method were used to detect 92 sheep serum samples,and the results showed that the positive coincidence rate was 81.6%,the negative coincidence rate was 92.6%,and the total coincidence rate was 88.0%.【Conclusion】 This indirect ELISA method could be used to detect clinical samples,and providing a reliable method for the evaluation of vaccine immune effect and disease diagnosis of Mycoplasma ovipneumoniae.

【Objective】 The purpose of this study was to express P[1] Bovine rotavirus A (BRVA) VP8 recombinant protein by prokaryotic expression system,and evaluate its reactivity and immunogenicity.【Method】 The codon preference was optimized according to the VP8 gene sequence of domestic epidemic strain RVA/Cow-tc/CHN/SDA2/2018/G6P[1] (GenBank accession No.:MN937517),the prokaryotic expression system of P[1] BRVA VP8 E.coli was constructed,the recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,the expression was induced by IPTG,and the recombinant protein was purified by nickel agarose affinity chromatography.Western blotting was used to identify the specific recognition ability of recombinant protein and rabbit anti-G6P[1] BRVA serum,and P[1] BRVA VP8 recombinant protein was used to immunize rabbits.Two weeks after the first immunization,the immunization was strengthened once and twice in total.The immunogenicity was evaluated by indirect ELISA and neutralization test.【Result】 The prokaryotic expression system of P[1] type BRVA VP8 recombinant protein was successfully constructed,SDS-PAGE showed that the recombinant protein was highly expressed with a size of 20 ku,and the recombinant protein existed in soluble form.Western blotting results showed that the recombinant protein could specifically bind to rabbit anti-G6P[1] BRVA serum.Indirect ELISA results showed that P[1] type BRVA VP8 recombinant protein could induce antibody production in rabbits.The antibody began to appear and increased rapidly in the 1st week after the first immunization,peaked in the 3rd week and remained at a high level until to the 5th week.The neutralization test results showed that the neutralization titers of immunized rabbit serum to G6P[1] and G8P[1] BRVA were 1∶17 783 and 1∶64,respectively.【Conclusion】 This study successfully constructed a prokaryotic expression system to express P[1] BRVA VP8 recombinant protein,and confirmed that it had good reactivity and immunogenicity,which laid a foundation for the development of BRVA subunit vaccine.

【Objective】 This study was aimed to understand the distribution and pathogenicity of Escherichia coli from ducks in Jiangsu,Jiangxi and Anhui provinces.【Method】 This study isolated and identified duck derived Escherichia coli from dead ducks in Jiangsu,Jiangxi and Anhui,determined the serotype of duck derived Escherichia coli isolates by PCR combined with slide agglutination,detected 18 virulence genes by PCR,and then carried out the pathogenicity test of ducklings.The growth curve and median lethal dose (LD₅₀) of the strains with strong and weak virulence were determined.【Result】 A total of 74 strains of duck derived Escherichia coli were isolated and identified in this study,1,2,2 and 4 strains were identified as O1,O2,O18 and O78 serotypes respectively,and the rest were not finalized.The identification test of 18 virulence genes showed that positive rates of genes ibeB,yijp,ompA and mat were 97.3%,97.3%,95.95% and 90.54%,respectively.Animal pathogenicity tests showed that 74 strains of Escherichia coli isolates were pathogenic to ducklings at infection dose of 10⁷ CFU/duck,but only 2 strains of isolates caused the death rate of ducklings ≥50%.The growth curve detemination showed that there was no significant difference between 2 strains of virulent isolates and 2 strains of attenuated isolates (P>0.05),and the LD₅₀ of 2 strains of virulent isolates were 10^4.75and 10^7.375 CFU,respectively.【Conclusion】 The rate of serotypes O1,O2,O18,O78 in 74 isolates was only 12.16%.The virulence gene spectrum was widely distributed,but only 2 strains of isolates were more virulent.This study laid the foundation for the prevention and control of Escherichia coli from ducks,it was also helpful for studying the relationship among serotypes,virulence genes and pathogenicity.

African swine fever (ASF) is an acute,febrile and highly contagious infectious disease caused by African swine fever virus (ASFV).Both morbidity and mortality rate of acute cases are almost 100%.ASF is recognized as a statutory report animal disease by the World Organization for Animal Health (OIE) and listed as class Ⅰ animal disease in China.At present,the situation of ASF epidemic prevention and control in China is very serious,and there is no effective vaccine and preventive and therapeutic agents for ASF.Therefore,strict sanitation and removal of infected animals through monitoring pathogen and antibody are important measures for control of ASF.In this paper,we summarized the recent progress in development of detection and serological monitoring methods for ASF built on its specific diagnostic marker.In etiological diagnosis,isothermal amplification techniques developed on the basis of PCR,such as recombinase polymerase amplification,crossing-priming amplification,loop-mediated isothermal amplification and dropletdigital PCR,overcome the defects of conventional PCR in different aspects.Real-time quantitative PCR provides fast,accurate,objective and quantitative results.The new CRISPR/Cas12a technology does not require an expensive instrument and offers a new,more portable,simple,sensitive and specific alternative to ASFV detection.On serological diagnosis,based on enzyme-linked immunosorbent assay (ELISA),immunochromatography and indirect immunofluorescence assay have also improved sensitivity and specificity.

Bovine hepacivirus,the novel member of the Hepacivirus,which was firstly discovered at bovine serum in Germany in 2015,has been found in 7 countries through high-throughput sequencing and has been divided into 2 genotypes and 8 subtypes,with at least 4 subtypes reported in China.Bovine hepacivirus whose hosts are member of Bovidae family,characterized by liver tropism,can cause acute or persistent infections in cattle,posing a potential threat to the development of the livestock industry and the health of related personnel.Bovine hepacivirus includes a genome of 8.8 kb,comprising 5'-UTR,3'-UTR and a long open reading frame (ORF) encoding 2 779 amino acids,and there is an internal ribosome entry site (IRES) associated with viral translation in 5'-UTR.Bovine hepacivirus,for which the main detection methods contain molecular biological diagnosis (nested-PCR) and immunological diagnosis (luciferase immunoprecipitation systems,LIPS),showing highly prevalent in cattle.However,with the increasingly development of emerging viruses,nested PCR may not detect Bovine hepacivirus with high genetic variation,and immunological tests may have cross-reactivity.So far,there is not only a lack of effective vaccines and other therapeutic drugs,but also a lack of awareness and measures of prevention and control towards Bovine hepacivirus in livestock farms.Therefore,this article of the latest research advances in the etiology,epidemiology,detection methods,prevention and control of bovine hepacivirus aims to increase awareness of Bovine hepacivirus,which can provide some reference and guidance for the research,prevention and control of the virus.

【Objective】The purpose of this experiment was to study the therapeutic effect of Wuwei Zhili mixture on diarrhea induced by Escherichia coli in mice.【Method】 Four-week-old clean grade ICR mice were divided into blank control (BC),model control (MC),Wuwei Zhili mixture high (W-H),medium (W-M),low (W-L) doses and ciprofloxacin hydrochloride control (CPFX) groups.20 mice in each group were intraperitoneally administered with Escherichia coli except BC group to construct the diarrhea model.When diarrhea symptoms appeared,the treatment group was intragastrically administered with high,medium and low doses of Wuwei Zhili mixture (25,20,15 mL/kg BW,equivalent to crude drug 2.5,2.0,1.5 g/kg BW),CPFX group was intragastrically administered with ciprofloxacin hydrochloride (20 mL/kg BW),and MC group and BC group were intragastrically administered with the same amount of normal saline,twice a day for 5 successive days.Diarrhea rate and protection rate were determined 14 days after drug withdrawal.Another 100 mice were divided and administrated according to the above methods,the duodenum and jejunum were collected on the days 2 after administration and the day 1,7 and 14 after drug withdrawal.The expressions of duodenal mucosa repair factor proliferating cell nuclear antigen (PCNA) and transforming growth factor-α (TGF- α) were detected by immunohistochemistry.【Result】 On the day 14 after drug withdrawal,the protection rates of diarrhea in W-H and W-M groups were 95% and 90%,and the mortality rates were both 0,which was similar to CPFX group.In the course of treatment,the small intestinal villi of mice in W-H and W-M groups gradually tended to be intact,the infiltration of lymphocytes gradually decreased,and the mucosal morphology gradually tended to be normal.The sIgA contents in W-H,W-M and CPFX groups were significantly higher than that in MC group (P<0.05).The secretion of IFN-α in intestinal mucosa of W-H,W-M,W-L and CPFX groups were significantly inhibited (P<0.05),and the secretion of IL-4 were significantly increased (P<0.05) when compared to that of MC group at day 1,7 and 14 after drug withdrawal.On the day 14 after drug withdrawal,the contents of IFN-α and IL-4 in W-H group were similar to that in BC group.On day 1 and 7 after drug withdrawal,the expression level of intestinal repair factor PCNA in W-H and CPFX groups was significantly higher than that in MC group (P<0.05).On day 1 after drug withdrawal,the expression level of intestinal repair factor TGF-α in W-H and CPFX groups was significantly higher than that in MC group (P<0.05),and compared with MC group,there was no significant difference in TGF-α expression in W-H and CPFX groups on day 7 after drug withdrawal (P>0.05).There were no significant differences in the expression of PCNA and TGF-α among all groups on day 14 after drug withdrawal (P>0.05).【Conclusion】 2.5 g/kg BW Wuwei Zhili mixture could significantly control diarrhea caused by Escherichia coli in mice,and the protection rate was 95%.It played a prominent therapeutic role by increasing the content of sIgA in jejunal lavage fluid,fully activating immune cells,regulating the cytokines IFN-α and IL-4 secreted by intestinal mucosa,effectively inhibiting intestinal inflammatory response,stimulating the expression of intestinal mucosal repair factors PCNA and TGF-α.