【Objective】 This study was aimed to clone,express and bioinformatics analyze the CDS region of goats interferon-stimulated gene 15 (ISG15) and investigate the effect of Peste des petits ruminants virus (PPRV) infection on ISG15 in caprine endometrial epithelial cells (EEC).【Method】 According to the prediction sequence of ISG15 gene of goats published in GenBank (accession No.:XM_005690795),specific primers were designed and RT-PCR was used to amplify the CDS region of ISG15 gene in goats which was connected to the eukaryotic expression vector for sequencing and expression.The nucleotide and amino acid sequences of ISG15 from different species were compared, and the phylogenetic tree was constructed. Physicochemical properties,transmembrane structure,modification sites,secondary structure,tertiary structure,and subcellular localization of ISG15 protein were analyzed by the bioinformatics method.EEC cells were transfected by constructing eukaryotic expression vectors and infected with PPRV.The exogenous and endogenous subcellular localization were observed by the indirect immunofluorescence method,and the effects of PPRV infection on EEC cells were investigated.【Result】 The CDS region of ISG15 gene in goats was successfully cloned and expressed in eukaryotes.The nucleotide and amino acid similarities and evolutionary tree analysis of ISG15 in goats were closest to Ovis aries and Ovis ammon.Bioinformatics analysis showed that the ISG15 gene in goats was located on chromosome 16 with a total length of 474 bp,encoding 157 amino acids,and a molecular weight of 17.47 ku. It was a hydrophilic protein without transmembrane region and signal peptide and had 1 potential N-glycosylation site,16 potential O-glycosylation sites,and 10 potential phosphorylation sites.The prediction of the secondary and tertiary structure showed that ISG15 protein was composed of the random coil,extended chain,alpha helix,and beta turn,accounting for 34.39%,31.21%,21.66%,and 12.74%,respectively.It could interact with interferon and ubiquitination-related proteins and participate in the body's anti-infection effects action.Subcellular localization showed that both exogenous and endogenous ISG15 were localized in the cytoplasm.【Conclusion】 The CDS region of ISG15 gene in goats was successfully cloned,and subcellular localization showed that both exogenous and endogenous ISG15 were localized in the cytoplasm,which laid a foundation for subsequent intracellular functional studies of ISG15 gene.

【Objective】 This study was aimed to screen the key genes of the difference of intramuscular fat (IMF) content of large Diqing Tibetan pigs (TPs) at different growth stages and analyze its regulation pathway.【Method】 There were thirty-six TPs with the same parity,date of birth and weight were randomly divided into three groups.The fattening test was carried out under the same conditions.They were slaughtered when the weight was about 40,80 and 120 kg,respectively.The longissimus dorsi muscle (LD) of three pigs in each group was collected,and the transcriptome was sequenced by RNA-Seq.The sequencing data was spliced,compared and annotated,the significantly differentially expressed genes (DEGs) related to IMF deposition were screened,and the gene interaction network was constructed by functional enrichment and STEM analysis.The results were verified by Real-time quantitative PCR.【Result】 There were 730,981 and 735 genes were significantly differentially expressed in 40 kg vs 80 kg,80 kg vs 120 kg and 40 kg vs 120 kg stages,respectively.There were four modules with significant differences in STEM analysis.The gene sets of modules 11 and 14 were significantly up-regulated and then slightly down-regulated.The gene sets of modules 9 and 10 were significantly up-regulated and then down-regulated.The differential gene interaction network showed that at 40 kg vs 80 kg stage,EGR1,EGR2,PRKAG2,NOR-1 and ATF3 genes were located in the core of the network,EGR1 and EGR2 genes were down-regulated,PRKAG2,NOR-1 and ATF3 genes were up-regulated.At 80 kg vs 120 kg stage,FOXO1,PDK4,PPARD,PPARGC-1,LIPE,ATF3 and STAT1 genes were located in the core of the network,FOXO1,PPARD and PPARGC-1 genes were down-regulated,and PPARG gene caused STAT1 gene down-regulation through cascade regulation,LIPE and ATF3 genes were up-regulated.At 40 kg vs 120 kg stage,ATF3,NOR-1,EGR1,EGR2 and STAT1 genes were located in the core of the network,EGR1,EGR2 and STAT1 genes were down-regulated,ATF3 and NOR-1 genes were up-regulated.Real-time quantitative PCR results of ten genes such as ATF3 and FOXO1 were consistent with RNA-Seq results.【Conclusion】 This study screened some genes like EGR1 and FOXO1 participate in the regulation of IMF in TPs as core genes,which involved in regulation at different stages were not exactly the same.The results could enrich the basic data of IMF regulation in local pigs in China,and provide a reference for the genetic improvement of IMF content in TPs.

【Objective】 The aim of this study was to explore the relationship between WIP1 gene and adipocyte proliferation and differentiation,in order to provide new genetic material for pig quality trait breeding.【Method】 Three pairs siRNAs of WIP1 gene (WIP1-790,WIP1-893 and WIP1-1845) and negative control NC-siRNA were transfected into 3T3-L1 preadipocytes by lipofection respectively.The expression level of WIP1 gene was detected by Real-time quantitative PCR to compare the interference efficiency of different siRNAs.Then the siRNA with the highest interference efficiency was used to interfere with the expression of WIP1 gene in 3T3-L1 preadipocytes.The cell proliferation of siRNA knockdown cells and NC-siRNA cells at different growth stages (0,12,24,48,72,96 and 120 h) was detected by CCK-8,and the expression of Cyclin B1 and Cyclin D1 genes at 24 and 48 h were also determinated by Real-time quantitative PCR.In addition,the 3T3-L1 preadipocytes of NC-siRNA and siRNA with the highest interference efficiency groups were induced to adipogenic differentiation.The efficiency of adipogenic differentiation was evaluated by oil red O staining and triglyceride quantification,respectively.The expression of peroxisome proliferator-activated receptor γ(PPARγ),CCAAT/Enhancer binding protein (C/EBPα),fatty acid binding protein 4 (FABP4),and stearoyl-coenzme A desaturase 1 (SCD1) were detected by Real-time quantitative PCR.The expression of PPARγ protein was detected by Western blotting.The temporal and spatial expression of WIP1 gene in inguinal white adipose tissues and perigonadal white adipose tissues of mice at different ages (21 days,8 weeks and 6 months) was detected by Real-time quantitative PCR.【Result】 The results of gene interference test showed that compared with NC-siRNA group the three pairs of siRNAs had an extremely significant interference effect on the expression of WIP1 (P<0.01),and the interference efficiency of WIP1-790 was the highest,reaching more than 70%.The CCK-8 test results showed that compared with NC-siRNA group,the proliferation rate of WIP1-790 interference group was extremely significantly decreased at all growth stages(P<0.01),and the expression of Cyclin B1 and Cyclin D1 genes were also significantly down regulated at 24 and 48 h (P<0.01).The results of adipogenic differentiation experiment showed that compared with NC-siRNA group,the oil red O positive cells were obvious reduced,the contents of lipid droplets in was extremely significantly decreased (P<0.01),and the content of triglyceride was significantly decreased (P<0.05) in WIP1-790 interference group on the 8 d of adipogenic differentiation.The mRNA expression levels of PPARγ,C/EBPα,FABP4 and SCD1 genes were extremely significantly lower in the WIP1 gene interfering cells than those in the negative control cells(P<0.01).The level of PPARγ protein in the WIP1 gene interfering cells was also extremely significantly lower than that of negative control cells(P<0.01).At the individual level of mice,the expression of WIP1 gene in inguinal white adipose tissues of 8-week-old and 6-month-old mice was extremely significantly higher than that of 21-day-old mice(P<0.05;P<0.01),and the expression of WIP1 gene in perigonadal white adipose tissues of 6-month-old mice was extremely significantly higher than that of 21-day-old and 8-week-old mice(P<0.01).【Conclusion】 The results revealed that WIP1 gene could affect the proliferation and differentiation of 3T3-L1 cells by regulating the expression of cell cycle and adipogenic differentiation-related genes such as Cyclin B1,Cyclin D1 and PPARγ,and it was also participated the process of fat deposition.This study would provide basic data for further analysis of the molecular mechanism of adipogenesis.

【Objective】 Using a single base editor to introduce a stop codon at the exon 2 of myostatin(MSTN) gene in Ningxiang pig,and obtaining kidney fibroblast cells with MSTN gene silencing,which was the foundation for the later breeding of MSTN base-editing pigs.【Method】 In this study,a single guide RNA (single guide RNA,sgRNA) was designed at the exon 2 of MSTN gene,and connected to the pMLM3636-puro plasmid to form a recombinant expression vector pMLM3636-puro-MSTN,co-transfected into kidney fibroblast cells with YE1-BE3-FNLS containing the red fluorescent base editor.Under the conditions of red fluorescence and puromycin,single cell colonies were picked.After confirmed by sequencing,the protein expression of the target mutant cell lines was analyzed.【Result】 Base site directed mutation occurred in the exon 2 of MSTN gene,the amino acid sequence of the target site was changed from tryptophan (TGG) to a stop codon (TAA),and the G→A mutation rate was 5.5%.The results of Western blotting showed that the MSTN protein expression in the experimental group No.10 single clone cells decreased by 60% compared with the wild type.【Conclusion】 In this study,single base editing technology was used to introduce stop codon in the coding region of MSTN gene of Ningxiang pig,so that translation was terminated early,resulting in a significant decrease in protein expression,which laid a foundation for the production of MSTN base mutant pigs.

【Objective】 This study was aimed to understand the codon usage characteristic and explore the evolution pattern of vitamin D receptor (VDR) gene in Ovis aries,and provide reference basis for establishing animal model related to bone metabolism diseases.【Method】 The relative synonymous codon usage (RSCU),codon composition,effective number of codons (ENc),codon bias index (CBI) and several parameters were analyzed using CodonW,EMBOSS,Usage Codon and DNAMAN softwares,and compared these parameters among multiple species,meanwhile,ENc-Plot and neutrality plot were used to analyze the main influencing factors of codon bias use of VDRgene.The internal relationship between codon use patterns and biological evolution was explored by means of relative synonymous codon usage of multiple species and cluster analysis.【Result】 There were 23 codons of VDRgene in Ovis ariespreferred to use codons ending with G/C.The other 14 species,such as Gorilla gorilla gorilla,Dromaius novaehollandiaeand Rattus norvegicusshowed similar characteristics in codon usage with Ovis aries.While,some species,such as Dromaius novaehollandiae,Rattus norvegicusand Passer montanus showed unique characteristics in the coding of Cys,Gly,His,Ser and Pro.ENc-Plot and neutrality plot analysis showed that selection pressure was stronger than mutation pressure on the codon usage bias of VDR gene.【Conclusion】 The codon ending with G/C was the dominant codon of VDR gene,the natural selection was the main force leading to the phenomenon.Meanwhile,the codon usage bias was species-specific,and there was a correlation with the evolution of genetic relationship.

【Objective】 This study was conducted to identify the major genes related to abdominal fat deposition in ileum of muscovy ducks.【Method】 200 70-day-old muscovy ducks were randomly selected from 2 000 muscovy ducks for slaughter.The abdominal fat was collected and weighed to calculate abdominal fat percentage.Sorted by abdominal fat rate,the ileum tissues of 5 muscovy ducks with the highest abdominal fat percentage and 5 muscovy ducks with the lowest abdominal fat percentage were selected for transcriptome sequencing analysis.Illumina NovaSeq 6000 high-throughput RNA-Seq sequencing technology was used to perform transcriptome sequencing on ileum of 2 kinds of muscovy ducks with abdominal fat rate.RSEM software was used to compare the sequenced reads sequence with the reference genome of Trinity to screen out differentially expressed genes.GO and KEGG databases were used for functional annotation,enrichment analysis and cluster analysis,and Pearson correlation analysis was used to analyze the correlation between abdominal fat weight and abdominal fat percentage and differentially expressed genes.【Result】 There were 602 differentially expressed genes in ileum of muscovy ducks with high and low abdominal fat rates.Compared with the muscovy ducks with low abdominal fat rate,285 genes were up-regulated and 317 genes were down-regulated in ileum of muscovy ducks with high abdominal fat rate.GO and KEGG enrichment analysis showed that there were 5 GO terms (lipid response,steroid hormone-mediated signaling,steroid hormone response,steroid hormone-stimulated cellular response,and lipid cellular response) and 4 KEGG pathways (PPAR signaling,biosynthesis of primary bile acids,arachidonic acid metabolism,and bile secretion) related to lipid metabolism.In addition,6 genes were found to be significantly correlated with fat metabolism (P<0.05),including NR5A2,ACBP,ACOX2,FABP2,FABP4 and FABP5,among which ACOX2 and FABP5 genes were significantly positively correlated with abdominal fat weight and abdominal fat percentage (P<0.05),ACBP gene was significantly positively correlated with abdominal fat weight and abdominal fat percentage (P<0.01).【Conclusion】 There were differences in the ileum transcriptome of muscovy ducks with different abdominal fat rates.It was found that 5 GO terms and 4 KEGG pathways,NR5A2,ACBP,ACOX2,FABP2,FABP4 and FABP5 genes were related to abdominal fat deposition in muscovy ducks.

【Objective】 The purpose of this study was to analyze the CDS sequence of myostatin (MSTN) gene and its expression in different growth stages and different tissues of Yangyuan donkeys.【Method】 Blood samples and various tissue samples were collected from Yangyuan donkeys aged 6,12,18 and 24 months old,genomic DNA and total RNA from different tissues were extracted. The CDS sequence of MSTN gene in Yangyuan donkeys was amplified by PCR and analyzed by bioinformatics.Real-time quantitative PCR was used to detect the expression of MSTN gene in longissimus dorsi muscle,leg muscle of Yangyuan donkeys at different growth and development stages, and different organizations (heart,liver,spleen,lung,kidney) of 18 months old Yangyuan donkeys.【Result】 The CDS sequence of MSTN gene in Yangyuan donkeys was 1 128 bp and encoded 375 amino acids,which had the highest similarity of nucleotide sequences (99.9%) with Equus caballus.The encoded protein was an unstable hydrophilic protein,containing transforming growth factor β (TGF-β)superfamily domain,no transmembrane region,1 signal peptide region,6 O-glycosylation sites,1 N-glycosylation site and 34 phosphorylation sites,mainly distributed in nucleus (39.1%),and the proportion of random coil in the secondary structure was the highest (50.93%).The expression of MSTN gene in longissimus dorsi muscle and leg muscle of Yangyuan donkeys at 18 months old were significantly higher than that of 6,12 and 24 months old (P<0.05).MSTN gene was expressed in different tissues of 18 months old Yangyuan donkeys.The expression in longissimus dorsi muscle was significantly higher than that in other tissues (P<0.05),followed by leg muscle,lung,spleen,kidney and liver,and the expression in heart was the lowest.【Conclusion】 The CDS sequence of MSTN gene in Yangyuan donkeys was successfully amplified.The expression of MSTN gene in longissimus dorsi muscle and leg muscle of Yangyuan donkeys at different growth and development stages was the highest at 18 months old,and the expression of longissimus dorsi muscle and leg muscle was higher in different tissues at 18 months old.The results provided a reference for further exploring the mechanism of MSTN gene in the growth and development of skeletal muscle in Yangyuan donkeys.

【Objective】 The objective of this study was to analyze the expression profile of Krüppel-like factor 7 (KLF7) gene,and explore the effect of its overexpression on proliferation and differentiation of preadipocytes in Tibetan sheep.【Method】 Preadipocytes that isolated from adipose tissue in Tibetan sheep were cultured and induced differentiation.The mRNA relative expression of KLF7 gene in 7 tissues (brain,subcutaneous fat,kidney,longissimus dorsi muscle,rumen,testis and ileum) and preadipocytes at different differentiation stages (0,2nd,4th and 8th days) in Tibetan sheep were analyzed by Real-time quantitative PCR.The CDS sequence of KLF7 gene was amplified from adipose tissue by RT-PCR,and ligated into the pcDNA3.1(+) eukaryotic expression vector to conduct pcDNA3.1-KLF7 overexpression plasmid.The pcDNA3.1-KLF7 overexpression plasmid was transfected into preadipocytes,and the mRNA expression of adipocyte proliferation and differentiation marker genes were detected by Real-time quantitative PCR.The EdU-positive cell,cell viability and production of lipid droplets in KLF7 gene overexpressing preadipocytes were detected by EdU,CCK-8 and Oil red O staining methods,respectively.【Result】 KLF7 gene was expressed in 7 tissues of Tibetan sheep,and the highest expression was found in brain,followed by subcutaneous fat and kidney,which were significantly higher than other tissues (P<0.05).The mRNA expression of adipocytes which was induced at the 2nd,4th and 8th days were significantly higher than that of undifferentiated cell (P<0.05),and the highest expression was identified in adipocytes at the 2nd day after differentiation.In KLF7 gene overexpressing preadipocytes at the 2nd day,the expression of CDK4,CyclinB1 and CyclinD1 genes were significantly or extremely significantly inhibited (P<0.05 or P<0.01),and the cell viability and the number of EdU positive cell were also extremely significantly decreased (P<0.01).In KLF7 gene overexpressing preadipocytes at the 8th day,the expression of PPARγ,Glut4 and ELOVL6 genes were significantly or extremely significantly down-regulated (P<0.05 or P<0.01),and the lipid deposition was extremely significantly reduced (P<0.01). The results indicated that KLF7 gene overexpression inhibited the proliferation and differentiation of preadipocytes in Tibetan sheep.【Conclusion】 The widely expressed of KLF7 gene was found in tissues of Tibetan sheep,and there was a higher expression in brain,subcutaneous fat and kidney.The expression of differentiated adipocyte was significantly higher than that of undifferentiated,and the highest expression was observed at the 2nd day after differentiation.KLF7 gene overexpression could inhibit the proliferation and differentiation of preadipocytes in Tibetan sheep,which provided basic data for elucidating the molecular regulation mechanism of fat deposition in Tibetan sheep.

Porcine skeletal muscle is an important movement organization of animal body,the main meat source of human beings,and a good model for muscle growth and disease research.After birth,pig needs the participation of muscle satellite cells in the growth and development of skeletal muscle,and the damage repair.Isolation and culture of porcine skeletal muscle satellite cells in vitro is the basis for in-depth study on the growth and development of skeletal muscle and the pathogenesis of diseases,and the premise for molecular function verification at the cellular level.With the deepening of the research on muscle development and pathological molecular mechanism,the technology of isolation and culture of satellite cells of porcine skeletal muscle in vitro has developed rapidly.The longissimus dorsi muscle,hind leg muscle and semitendinosus muscle are commonly used to isolate skeletal muscle satellite cells,and the longissimus dorsi muscle of 1-day-old piglets have the best isolation effect.Enzymes commonly used to isolate skeletal muscle satellite cells include pronase,collagenase,trypsin,collagenase,etc.,and the time of digestion of each enzyme and its combination is different.The best filtration method is 200 mesh +400 mesh combined filtration,and high purity cells can be obtained by three times centrifugation.The usual medium is DMEM/F12+10% fetal bovine serum (FBS)+1% penicillin-streptomycin (P/S).Common markers of skeletal muscle satellite cells include pairing box gene 3(PAX3),PAX7,Myo-genic determinant 5(Myf5),Myf4,Myo-differentiation factor (MyoD),myogenin (MyoG) and so on.In this paper,the isolation,culture and identification of muscle satellite cells of porcine skeletal muscle were reviewed,and the best parameters in each step were sorted out,which provided references for establishing a standardized procedure for isolating porcine skeletal muscle satellite cells,in order to provide theoretical and technical support for muscle development and disease research.

【Objective】 This experiment was conducted to investigate the effects of dietary metabolizable energy level on the growth performance,nutrient apparent metabolic rate,energy utilization and serum biochemical indexes,and to clarify the optimum dietary metabolizable energy level of male pheasants aged from 13 to 17 weeks.【Method】 A total of 240 healthy male pheasants at 13 weeks of age with similar body weight of (734.63 g±18.03) g were randomly assigned into 4 groups with 6 replicates per group and 10 pheasants per replicate.A one-factor design was adopted in this study,dietary metabolizable energy levels were set as 11.30 (group Ⅰ),11.72 (group Ⅱ),12.14 (group Ⅲ) and 12.56 (group Ⅳ) MJ/kg,respectively.The pre-trial period lasted for 7 days and the trial period lasted for 35 days.【Result】 ① The average daily feed intake (ADFI) were extremely significantly affected by dietary metabolizable energy levels (P<0.01).The ADFI in group Ⅳ was extremely significantly lower than that in groups Ⅰ and Ⅱ (P<0.01).Dietary metabolizable energy levels had no significant effect on feed to gain ratio (F/G),and it was the lowest in group Ⅱ (P>0.05).② Dietary metabolizable energy levels had extremely significant effects on dry matter (DM) and ether extract (EE) apparent metabolic rates (P<0.01).Crude protein (CP) apparent metabolic rate was not significantly affected by dietary metabolizable energy levels,and it was the lowest in group Ⅱ (P>0.05).③ Dietary metabolizable energy levels had on significant effect on gross energy intake,gross energy in excreta and metabolizable energy intake (MEI) (P>0.05).Gross energy apparent metabolic rate in groups Ⅱ, Ⅲ and Ⅳ were extremely significantly higher than group Ⅰ (P<0.01).④ The contents of serum cholesterol (CHO),low density lipoprotein cholesterol (LDL-C) and glucose (GLU) were not significantly affected by dietary metabolizable energy levels (P>0.05).The content of triglyceride (TG) in group Ⅲ was extremely significantly higher than that in other groups (P<0.01),and the content of serum high density lipoprotein cholesterol (HDL-C) in group Ⅱ was extremely significantly higher than that in group Ⅰ (P<0.01).The contents of total protein (TP) in groups Ⅱ and Ⅲ were significantly higher than that in group Ⅰ (P<0.05),and the content of serum albumin (ALB) in group Ⅲ was significantly or extremely significantly higher than that in other groups (P<0.05 or P<0.01).【Conclusion】 Considering all the factors,the pheasants in group Ⅱ could get better average daily gain (ADG),F/G,higher apparent metabolic rates of nutrients,higher energy intake and energy utilization.The dietary metabolizable energy level in group Ⅱ (measured value was 12.30 MJ/kg) was suitable for male pheasants aged from 13 to 17 weeks.

【Objective】 The effects of dietary condensed tannins (CT) on growth performance,body composition,apparent digestibility of nutrients,and intestinal morphology of Lateolabtax maculatus were studied to assess the potential application value of CT as aquatic feed additive.【Method】 A total of 480 Lateolabtax maculatus with an initial body weight of (2.76±0.05) g were randomly divided into 4 groups with 4 tanks per group and 40 shrimp per tank.Three isonitrogenous and isolipidic diets,named T1 (basal diet,control group),T2 (basal diet+1 g/kg of CT) and T3 (basal diet+2 g/kg of CT) were prepared in this study,and fish was fed to apparent satiation during the 63-d feeding trial.【Result】 Survival rate and feed conversion ratio were similar among 4 groups (P>0.05). The final body weight,weight gain rate and specific growth rate were lower in T3 group than those in T1 and T2 groups (P<0.05).Dietary treatment did not alter the whole body composition of moisture,crude protein,crude lipid and ash (P>0.05).Apparent digestibility of dry matter and crude protein were T1>T2>T3 (P<0.05). The apparent digestibility of crude lipid was lower in T3 group than that in T1 group (P<0.05),but it was similar between T1 and T2 groups (P>0.05).Dietary treatment did not alter the apparent digestibility of ash (P>0.05).Compared with T1 group,Lateolabtax maculatus in T2 and T3 groups had different degree of intestinal injury along with decreased villus length (P<0.05) and increased goblet cell numbers (P<0.05).【Conclusion】 Adding 1 g/kg of CT did not alter growth performance of Lateolabtax maculatus,but reduced apparent digestibilities of dry matter and crude protein.Adding 2 g/kg of CT inhibited growth performance,decreased nutrients apparent digestibility and damaged intestinal morphology of fish.CT could be used as feed additive to be applied in Lateolabtax maculatus diet,and the dosage should be less than 1 g/kg.

【Objective】 This experiment was conducted to investigate the effects of steam-flaked corn on the weight gain,rumen fermentation and microbial community of Xinjiang Brown cattle, and to explore the feeding methods to improve the weight gain performance and feeding efficiency of beef cattle.【Method】 Sixteen healthy male Xinjiang Brown cattle with similar age (18-month-old) and body weight (436.87 kg±17.29 kg) were randomly divided into 2 groups with 8 calves per group.The steam-flaked corn was used in the concentrate of experimental group and the chopped corn was used in control group.A 15-d adaptive phase was followed by 180-d of experimental period.Initial body weight and final body weight were recorded and average daily gain was calculated.The rumen fluid of Xinjiang Brown cattle was collected at 0,1,3,5,7,9 h after feeding on the 180 th day of the experiment to determine the rumen fermentation indexes,and the rumen fluid was collected at 0 h to analyze the composition of rumen microbial flora by 16S rRNA.【Result】 Compared with the control group,the average daily gain was significantly increased (P<0.05),the pH of rumen fluid was significantly decreased at 1 h (P<0.05) and extremely significantly decreased at 3 h (P<0.01), the contents of acetic and acetic+propionic+butyric in rumen fluid were significantly increased (P<0.05),and the content of propionic was significantly increased (P<0.01), ammonia nitrogen and microbial protein in rumen fluid increased significantly at all sampling time points (P<0.01) of the experimental group.There was no significant difference in Chao1 index and Shannon index between the two groups (P<0.05).Compared with the control group, at the phylum level,the relative abundance of Bacteroidetes was significantly increased (P<0.05),and the relative abundance of Spirochaetota was significantly increased (P<0.01),however,the relative abundance of Firmicutes,Desulfobacterota and Verrucomicrobiota decreased significantly (P<0.05),and the relative abundance of Actinobacteriota decreased very significantly (P<0.01) in the experimental group. At the genus level,the relative abundance of Rikenellaceae_RC9_gut_group,Prevotellaceae_UCG-003,Lachnospiraceae_XPB1014_group and Lachnospiraceae_NK3A20_group in the experimental group increased significantly (P<0.05),while the relative abundance of Christensenellaceae_R-7_group decreased significantly (P<0.05).【Conclusion】 The utilization of steam-flaked corn in diet could significantly increase the average daily gain,change the ruminal fermentation speed,improve fermentation efficiency, increase the microbial abundance related to carbohydrate and volatile fatty acid metabolism,and reduce the microbial abundance related to fiber degradation and disease of Xinjiang Brown cattle.

【Objective】 The aim of this study was to evaluate the genetic parameters of reproductive and growth traits in Duroc(DD),Landrace(LL) and Yorkshire (YY) pigs,and analyze the genetic trend changes of breeding values in different years,so as to provide a theoretical foundation for optimizing breeding programs.【Method】 The data of reproductive and growth traits in DD,LL and YY were used as the research materials.The 10 963 reproductive trait records,including total number born (TNB),number born alive (NBA),litter born weight (LBW) and litter weight at 21 days (LW21),and 25 257 growth trait records,including age at 100 kg live weight(AGE) and backfat adjusted to 100 kg(BF),were used in this study.The best linear unbiased prediction(BLUP) method based on animal model was used to estimate heritability,genetic correlation and breeding values by ASReml package.【Result】 The heritability of TNB,NBA and LBW ranged from 0.08 to 0.20,and the heritability of LW21 ranged from 0.02 to 0.05.The heritability of AGE and BF traits ranged from 0.22 to 0.37.The genetic correlation coefficients of reproductive traits of TNB,NBA,LBW and LW21 ranged from 0.20 to 0.97,which showed moderately positive correlations.The genetic correlation coefficients of AGE and BF ranged from -0.07 to -0.03,which showed slightly negative correlations.The genetic trend of reproductive traits of DD increased significantly,while that of LL and YY increased slightly.Among the growth traits,the genetic trends of AGE changed significantly,While the genetic trends of BF were varied less.【Conclusion】 This study accurately evaluated the genetic parameters and genetic trends of reproductive and growth traits of DD,LL and YY,the results could provide reference for the breeding work of the breeding field.

【Objective】 This study was aimed to reveal the genomic patterns of the phenotypic convergence of important economic traits in terminal sire Pietrain and Duroc pigs under artificial selection.【Method】 Based on the 50K SNP chips data of 376 Pietrain pigs,451 Duroc pigs line Ⅰ,841 Duroc pigs line Ⅱ and 497 Duroc pigs line Ⅲ,the integrated haplotype score (iHS) and allele frequency difference (△AF) were calculated in 100 kb windows with a step size of 50 kb,and the top 5% statistics of each method were taken as the putative selection signatures of ingroup and intergroup,respectively.The close selection signatures within the distance of 200 kb were merged by bedtools.The overlapping putative selection regions of iHS and △AF statistics between every two populations were defined as the genomic regions associated with trait convergence,and the parallel selection signatures related to important economic traits of pigs were explored based on these putative regions.【Result】 The results showed that 5 112 selection signatures with a total length of 487.51 Mb and 9 579 selection signatures with a total length of 913.50 Mb were detected by iHS and △AF,respectively.After merging the close region,a total of 52 parallel selection regions with the length of approximately 4.67 Mb were found,88 candidate genes were annotated to be related to the traits such as reproduction,carcass and meat quality.【Conclusion】 A total of 52 genomic selection regions might be associated with trait convergence between Pietrain and Duroc pigs,the parallel selection signatures were mainly involved in economically important traits such as reproduction,carcass and meat quality,which was related to the similar breeding direction of lean pigs,and the identification of these key genes could provide a reference for the genetic improvement of subsequent commercial pig breeds.

【Objective】 The analysis of population genetic structure,selection signal analysis and ROH detection were performed to Sunan yak to explore the genes related to the germplasm characteristics of Sunan yak.【Method】 A total of 48 yaks were collected from Sunan yak,Bazhou yak,Sibu yak,Jiulong yak and Tianzhu White yak for genome resequencing.Genome Analysis Toolkit (GATK) mutation detection process was used to obtain high-quality single nucleotide polymorphism (SNP) for downstream analysis.Principal component analysis,ancestral component analysis and phylogenetic tree analysis were used to determine the population structure of 5 yak breeds.ROH detection and inbreeding coefficient calculation of 5 yak populations were carried out.A comprehensive haplotype score (iHS) was used to screen selected sites in shared high-frequency ROH regions.【Result】 By analyzing the SNPs identified from 5 yak breeds,a total of 15 092 883 SNPs were identified.The identified SNPs were mainly distributed in the intron regions.The results of kinship calculation showed that all individuals did not have kinship within 3 generations,which met the requirements of subsequent analysis.Principal component analysis showed that the 5 yak breeds could be significantly divided into 5 groups,and the genetic variation within the Sunan yak group was small.The population structure showed that Sunan yak contained other 4 yak ancestors,among which Tianzhu White yak accounted for the largest proportion.ROH analysis showed that a total of 8 426 ROH fragments were detected in 5 yak breeds,of which 421 were in high-frequency ROH regions.Compared with the other 4 yak breeds,Sunan yak had the largest total length and number of ROH regions and had a higher degree of linkage,and its inbreeding coefficient was much larger than that of other yak populations.465 candidate genes were annotated on the high-frequency ROH region of Sunan yak,mainly enriched in pathways related to body development,brain morphogenesis,and fat oxidation,including embryonic skeletal system morphogenesis,anterior and posterior pattern specification,thyroid development pathways and Hippo path.Among them,genes related to embryonic development and tissue differentiation (homeobox A3 (HOXA3),HOXA5,HOXD3),meat quality (arachidonic acid 12B (ALOX12B),ALOX15B,ALOXE3),and midbrain development (fibroblast growth factor 8 (FGF8)) were identified.In addition,3 shared genes (heterogeneous nuclear ribonucleoprotein K (HNRNPK),kinesin family member 27(KIF27),G kinase anchor protein 1 (GKAP1)) were identified in a high-frequency ROH region on chromosome 7 (Chr7:12 661 870-13 045 935),which were related to the plateau adaptation shared by yak.The iHS analysis of loci within the shared high-frequency ROH region identified selected genes that were mainly associated with disease resistance,endoplasmic reticulum-secreted protein processing,and cell cycle regulation,including N-alpha-acetyltransferase 25 (NAA25),endoplasmic reticulum protein 29 (ERP29),transmembrane protein16 (TMEM116),TRAF-type zinc finger domain containing 1 (TRAFD1) and HECT domain E3 ubiquitin protein ligase 4 (HECTD4) genes.【Conclusion】 This study systematically investigated the genetic diversity and identified candidate genes related to traits of Sunan yak at the genome-wide level,providing an important theoretical basis for the development and utilization of Sunan yak germplasm resources.

【Objective】 This study was performed to explore the polymorphism of fibroblast growth factor receptor 2 (FGFR2) gene in sika deer and its effect on antler weight traits.【Method】 All exons of FGFR2 gene of sika deer were sequenced and analyzed by direct sequencing.314 24-month-old sika deer were genotyped and haplotyped by MassARRAY^® SNP typing technology,and the correlation between different genotypes and haplotypes of FGFR2 gene and antler weight traits was analyzed.【Result】 A total of 12 SNPs were found in the FGFR2 gene in sika deer,of which 5 were located in the exon region,and none of the mutations caused amino acid changes,belonging to synonymous mutations,and the other 7 SNPs were located in the intron region.The typing results showed that g.80975864 T>G site was not successfully typing,and the remaining 11 SNPs were subjected to subsequent analysis.The 3 SNPs of g.80943673 T>C,g.80943683 C>A and g.80938352 C>T belonged to moderately polymorphic (0.25<PIC<0.5),and the rest belonged to low-polymorphism (PIC<0.25). χ² fitness test results showed that the 2 SNPs of g.80998742 G>A and g.80987708 G>A deviated from Hardy-Weinberg equilibrium (P<0.05),and the others were in Hardy-Weinberg equilibrium.(P>0.05).The results of association analysis showed that there was no significant difference in antler weight among the genotypes of the 11 SNPs (P>0.05).Haplotype results showed that there were five haplotypes in FGFR2 gene,and there was no significant difference between different haplotypes and antler weights (P>0.05).【Conclusion】 The 11 loci at FGFR2 were not the potential markers for antler weight.

【Objective】 The aim of this study was to screen candidate genes under selection associated with egg laying traits in Yili and Hortobágy geese using selection signal analysis.【Method】 Twenty-four adult Yili geese and Twenty-four adult Hortobágy geese with good health status and consistent feeding and management level were selected in this experiment.Blood samples were collected from the geese,genomic DNA was extracted,and whole genome resequencing technology was used for selection signal detection and analysis to select the candidate regions under selection,localized to the annotated genes,and then GO function and KEGG pathway enrichment analysis were performed to further screen the candidate genes related to laying traits in goose.【Result】 The average sequencing depth of whole genome resequencing was 15.28×,and the contrast rate with the reference genome was more than 97.31%.These two populations were analyzed by population differentiation index (Fst),and a total of 1 231 candidate regions were identified.After combined analysis with nucleotide diversity (Pi),a total of 10 candidate regions were selected from Yili geese population,and 5 annotated genes were obtained;A total of 353 candidate regions were selected from Hortobágy geese population,and 263 annotated genes were obtained.GO function and KEGG pathway enrichment analysis results showed that 6 candidate genes (BMP2,BMP6,MIS,ENO1,LIF,EP300) might be related to egg-laying traits in geese were initially selected,and IL-18 gene was also found to be possibly related to the avian immunity.【Conclusion】 The results of this study screened 6 candidate genes that might be associated with egg-laying traits in geese,and provided a bioinformatic reference for revealing the molecular regulatory mechanisms of egg-laying traits in geese.

【Objective】 This study was aimed to explore the effect of structural variation of chromodomain-helicase-DNA-binding protein 3 (CHD3) gene on the expression of CHD3 gene in different pig breeds, and preliminarily analyze the relationship between the CHD3 gene structural variation and the formation of wrinkle skin in the Xiang pigs.【Method】 In this study, ordinary Xiang pigs, Xiang pigs with wrinkle skin and Large White pigs were selected as the research subjects. UCSC, miRBase, miRanda and RBPsuite softwares were used to predict the repeated elements, miRNA binding sites and RNA binding protein (RBP) binding sites contained in the CHD3 structural variation interval. PCR method was used for genotyping of structural variation. Excel was used to calculate the population genotype frequency and allele frequency, and χ² test was used to analyze whether the genotype was in Hardy-Weinberg equilibrium. The effect of the variation on CHD3 gene expression was detected by Real-time quantitative PCR and Western blotting.【Result】 Bioinformatics analysis showed that the 3'-flanking region of CHD3 gene was a 254 bp short-scatter element (SINE), belonging to tRNA family, located in Chr12:53 115 742-53 115 995, which contained 35 miRNA binding sites and 10 RBP binding sites. Genotyping results showed that II genotype (insertion), ID genotype (heterozygous) and DD genotype (double deletion) were detected in all three pig groups, indicating that 264 bp structural variation in the 3'-flanking region of CHD3 gene was highly polymorphic in pigs. Genotype population distribution frequency showed that the frequency of D allele in Xiang pigs with wrinkle skin was extremely significantly higher than that in ordinary Xiang pigs and Large White pigs (P<0.01); χ² test showed that the structural variation of CHD3 gene was in Hardy-Weinberg equilibrium state in Xiang pigs with wrinkle skin and ordinary Xiang pigs (P>0.05), but unbalanced in Large White pigs (P<0.05). Real-time quantitative PCR and Western blotting analysis showed that the mRNA and protein expression of DD and ID genotypes of CHD3 in skin of Xiang pigs were extremely significantly higher than that of II genotype (P<0.01).【Conclusion】 The 264 bp structural variation in the 3'-flanking region of CHD3 gene was a SINE element with regulatory effect, which could promote the metabolism of CHD3 gene mRNA and post-transcriptional translation inhibition through the binding of RNA binding protein and miRNAs to the element, thus regulating the expression of CHD3 gene. The structural variation resulted in abnormal expression and accumulation of CHD3 gene, which might be related to the formation of wrinkled skin in Xiang pigs.

【Objective】 The aim of this study was to investigate the mechanism of dihydrotestosterone (DHT) involved in the regulation proliferation and the expression of anti-Müllerian hormone (AMH) of mouse granulosa cells.【Method】 Kunming mice(3 weeks)were injected with pregnant horse serum gonadotropin (PMSG,10 IU/mouse) to obtain granulosa cells.After granulosa cells was passaged for 48 h,the morphology of granulosa cells was identified by HE staining,the growth curve of granulosa cells was drawn,and the expression of follicle stimulating hormone receptor (FSHR) was identified by immunofluorescence.When the confluence of the second generation granulosa cells reached 50%,they were starved with serum-free DMEM/F12 medium for 12 h,and different concentrations of DHT (0,10^-9,10^-8,10^-7,10^-6,10^-5 mol/L) were added to the medium.After 48 h of culture,the proliferation of granulosa cells was detected.The expression of AMH gene and AMH protein were measured by Real-time quantitative PCR and ELISA,respectively. 10^-6 mol/L flutamide (AR specific inhibitor),10^-7 mol/L DHT,10^-7 mol/L DHT and 10^-6 mol/L flutamide,10^-5 mol/L DHT,10^-5 mol/L DHT and 10^-8 mol/L 11 ketodihydroprosterone (AR specific agonist) were added to the medium of granulosa cells,which were recorded as F,D7,DF,D5 and DK groups respectively,and the control group was not added with drugs.After 48 h of culture,the proliferation of granulosa cells and the content of AMH protein were detected.【Result】 Mouse granulosa cells cultured in vitro were spindle or paving stone shaped,and the growth curve was S-shaped.The cells were generally expressed FSHR.Compared with 0 mol/L DHT group,the cell proliferation of 10^-8 and 10^-7 mol/L DHT group were significantly and extremely significantly increased(P<0.05;P<0.01),and that of 10^-5 mol/L DHT group was decreased significantly (P<0.05). The relative expression of AMH gene and the content of AMH protein in 10^-7 mol/L DHT group were extremely significantly increased (P<0.01).Compared with D7 group,the proliferation of granulosa cells and the content of AMH protein in DF group were extremely significantly decreased (P<0.01).Compared with D5 group,the proliferation of granulosa cells and AMH protein in DK group were extremely significantly increased (P<0.01).【Conclusion】 10^-7mol/L DHT could significantly promote the proliferation and AMH expression of granulosa cells,while 10^-5 mol/L DHT significantly reduced the proliferation and AMH expression.Moreover,AR could mediate DHT to regulate proliferation and AMH expression of granulosa cells.

【Objective】 The purpose of this study was to explore the relationship between the polymorphisms of follicle stimulating hormone receptor (FSHR) gene and egg-laying traits,egg quality and hatching performance of Baicheng You chickens,so as to provide new molecular markers for the breeding of high-performance breeders.【Method】 Taking 129 Baicheng You chickens as the research objects,the SNPs were screened by DNA mixed pool resequencing technology,and the related loci were genotyped by Sequenom time-of-flight mass spectrometry technology,and the correlation between the polymorphism of FSHR gene and production performance of Baicheng You chickens was analyzed by the general linear model of SPSS 26.0 software.【Result】 There were two SNPs in exon 1 of FSHR gene:SNP1 (g.7640519 C>A)、SNP2 (g.7640639 T>C),and one SNP in exons 4 and 5:SNP3(g.7692270 C>T) and SNP4(g.7698478 A>G),there were three SNPs in exon 10:SNP5 (g.7716725 G>C),SNP6(g.7715900 A>G) and SNP7 (g.7716470 T>C).With the exception of SNP6,the detection rates were above 90%.Only two genotypes (CC and CA) were present at SNP1,with CC being the dominant genotype,three genotypes (TT,TC,and CC) were present at SNP2,SNP3 and SNP7,three genotypes (AA,AG and GG) were present at SNP4,three genotypes (CC,CG and GG) were present at SNP5. The results of polymorphism analysis showed that the age of first egg of CC genotype at SNP2 was extremely significantly higher than that of TT genotype (P<0.01), and significantly higher than that of CT genotype (P<0.05); The egg weight of TT genotype was significantly higher than that of CT genotype (P<0.05), and the dead embryo rate of setting eggs was significantly higher than that of CC genotype (P<0.05). The shell thickness of TT genotype at SNP3 was significantly higher than that of CC genotype (P<0.05). The total egg mass at 300 days old of CG genotype at SNP5 was extremely significantly higher than that of CC and GG genotypes (P<0.01), the average egg weight at 300 days old and weiht at first egg were significantly higher than that of GG genotype (P<0.05), the body weight at first egg was significantly higher than that of CC genotype (P<0.05), and the shell thickness of GG genotype was significantly lower than that of CC and CG genotypes (P<0.05). The weight at first egg and individual egg weight of TT genotype at SNP7 were significantly higher than that of CC and CT genotypes (P<0.05), the albumen height and yolk weight were significantly higher than that of CT genotype (P<0.05), and the Haugh unit values were extremely significantly higher than that of CC and CT genotypes (P<0.01).【Conclusion】 The CG genotype at SNP5 (g.7716725 G>C) of FSHR gene could be used as the marker genotype that affected the egg-laying performance of Baicheng You chickens,and TT genotype at SNP7 (g.7716470 T>C) of FSHR gene could be used as the dominant genotype that affected the eggs quality of Baicheng You chickens.

【Objective】 The purpose of this study was to locate the polled intersex syndrome (PIS) reproductive defect gene and analyze its genotypes in Laoshan dairy goats.【Method】 A total of 155 Laoshan dairy goats were used in this study,including 9 horned rams,34 horned ewes,29 hornless ewes,47 hornless rams and 36 intersex goats.Phenotypic characteristics of 36 intersex goats were analyzed.Blood genomic DNA of 155 Laoshan dairy goats was used for genetic sex identification. Three pairs of primers for PIS wt-2,PIS wt-3 and PIS var-2 were designed according to the complete sequence information of goat genome DNA (RefSeq:NC_030808.1).PCR was used to verify whether there were 198 bp substitution and 108 bp deletion in PIS region of Laoshan dairy goats,and the genotypes of PIS gene in goats with different traits were analyzed.【Result】 Of the 36 intersex goats,17 goats displayed male pseudointersex with large clitoris,12 goats were pseudofemale pseudointersex,while 7 goats showed short penile intersex,and their sex chromosome were XX. The PIS region of Laoshan dairy goats was completely absent,and there was no 198 bp substitution or 108 bp deletion in 129 427 003-129 427 905 bp region. The genotypes of intersex goats were mutation homozygous,and the mutation type was with 10.1 kb deletion/480 kb duplication double mutations.The genotype of all horned goats was wild-type homozygous without 10.1 kb deletion/480 kb duplication.Among the hornless goats,ten 10.1 kb deletion/480 kb duplication double mutation homozygous rams were identified,and the rest of the hornless goats were heterozygous with 10.1 kb deletion/480 kb duplication single mutation.【Conclusion】 The appearance of intersex Laoshan dairy goats was caused by a total deletion of 10.1 kb and a reverse insertion of a duplicated fragment about 480 kb in size,and the intersex trait only occurred in mutation homozygous ewes,while the intersex trait did not occur in rams with homozygous deletions.The results could provide a reference for further revealing genetic mechanisms of hornless traits and breeding new hornless lines from Laoshan dairy goats.

【Objective】 The aim of this study was to explore the expression patterns of amino acids,glucose and fatty acid transporters in porcine placenta at different gestation periods.【Method】 15 Duroc sows with similar genetic background and litter size were randomly divided into 3 groups.After estrus,all sows were artificially inseminated with semen collected from the same Duroc boar.The uteruses of sows in each group were taken out by anesthesia on day 40 (D40),65 (D65) and 95 (D95) of gestation.The uterus was quickly opened to separate the placental tissue of each fetus and the total RNA of placental tissue was extracted and synthesized cDNA.The synthesized primers were used for general PCR amplification and 2.0% agarose gel was used to detect the amplification products.Real-time quantitative PCR was used to detect and compare the relative mRNA expression levels of amino acid,glucose,fatty acid transporters associated genes in placenta at three pregnancy stages.【Result】 PCR results showed that the length of amino acid transporter-related genes(SLC7A1,SLC7A2,SLC7A3,SLC7A4,SLC7A10,SLC1A3,SLC1A5,SLC38A10,SLC36A1),glucose transporter related genes(SLC2A1,SLC2A2,SLC2A3,SLC2A10,SLC2A12,SLC2A13),and fatty acid transporter-related genes (FATP1,FATP2,FATP3,FATP4,FABP3,FABP7,CD36) were consistent with the expected.Real-time quantitative PCR results showed that in amino acid transporters,the expression of SLC7A4,SLC7A10 and SLC38A10 genes in D65 placenta were significantly higher than those in D40 placenta (P<0.05),while SLC7A2 gene was significantly lower than that in D40 placenta (P<0.05),and the expression SLC1A3 and SLC7A4 genes in D65 placenta were significantly lower than those in D95 placenta (P<0.05).In glucose transporters,the expression of SLC2A3 and SLC2A13 genes in D65 and D95 placenta were significantly higher than those in D40 placenta,while the expression of SLC2A1,SLC2A2 and SLC2A12 genes in D95 placenta were significantly lower than those in D65 placenta (P<0.05).In fatty acid transporters,the expression of FATP2,FATP4,FABP3,FABP5,FABP7 and CD36 genes in D65 placenta were significantly higher than those in D40 placenta(P<0.05),while the expression of FATP1, FATP4 and CD36 genes were significantly lower than those in D95 placenta (P<0.05).【Conclusion】 The placental SLC7A10,SLC38A10,SLC7A4,SLC2A3,FATP1,FATP4,FABP7 and CD36 might be nutrition transport-related genes affecting fetal growth and development in porcine pregnancy.

Prostaglandin F_2α (PGF_2α) is a luteinolytic factor,which is widely used in estrus synchronization and induced parturition in sows.In recent years,new functions of PGF_2α have been found one after another.As a pregnancy recognition signal of mammals,endogenous PGF_2α can bind to PGF_2α receptor (FP) in the fetus and endometrium,initiate downstream signal pathways,and play an important role in early embryonic development,implantation and pregnancy maintenance.Lack or insufficient synthesis will lead to pregnancy interruption or abortion.In view of the promoting effect of PGF_2α on the reproduction activities of female animals,the veterinary drug center has developed a variety of PGF_2α drugs,such as sodium chloroprostol,dinoprostaglandin F_2α and so on,which are widely used to start delivery and promote estrus.According to the optical activity of sodium chloroprostol,it can be divided into D-type and L-type enantiomers,in which D-type structure is the main active component of sodium chloroprostol.Exogenous PGF_2α drugs can not only induce simultaneous estrus and synchronous delivery of sows,promote postpartum health and increase the estrus rate of weaned sows,but also improve sow lactation ability and colostrum quality,improve piglet vitality and other production traits.This paper reviews the research progress of new functions of PGF_2α at home and abroad in recent years,including the dual effects of PGF_2α on periodic corpus luteum,the important effects of PGF_2α on early embryo development,implantation and late pregnancy delivery,as well as the important effects of PGF_2α on sow lactation and piglet vitality.It also summarizes the effects of several PGF_2α and its analogues on reproductive performance of female animals.The purpose of this study is to provide theoretical basis for the rational use of PGF_2α and its analogues.

【Objective】 The aim of this study was to construct a synthetic peptide vaccine based on protein P72 in African swine fever virus (ASFV) strain CAS19-01/2019(GenBank accession No.:MIN172368.1),and evaluate its immune efficiency by immunizing mice.【Method】 The physicochemical properties and structural information of P72 protein were analyzed by programs of ProtParam and SOPMA.The significant epitopes in T and B lymphocytes were screened by ABCpred,SVMtrip and IEDCB.The synthesized peptides were injected intramuscularly into mice with Freund's adjuvant.Antibodies against the synthesized peptides,T lymphocyte subsets,lymphocyte proliferation,cytokines contents of interleukin 4(IL-4),IL-2,interferon-γ (IFN-γ) and immunoglobulin (IgG) were detected using serum and other tissue from the immunized mice,and the immune efficacy of the synthetic peptide was evaluated from the perspectives of humoral immunity and cellular immunity.【Result】 Comprehensive analysis showed that P72 protein was a stable hydrophilic protein.In secondary structure,alpha helix,beta turn,extended strand and random coil accounted for 19.35%,5.42%,25.08% and 50.15%,respectively.Eight dominant epitopes of protein P72 were selected by comprehensive software analysis,including T cell epitopes 626-634,520-528,298-306,203-211 amino acids,and B cell epitopes 587-606,232-251,110-129,39-58 amino acids. Two peptides named P72-1 and P72-2 were synthesized by integrating the dominant epitopes together.The specific antibodies against the synthesized peptides P72-1 and P72-2 were determined in serum of mice after 14 days of the first immunization,reached the highest value on the 28th day after the first immunization,and highest antibody titers were 1:25 600 and 1:12 800,respectively.The value of CD4⁺/CD8⁺ of T lymphocyte subsets in immunized mice were significantly increased (P<0.05).The spleen lymphocyte proliferation test showed that the number of lymphocytes was enhanced in both groups (P<0.01).Cytokines contents of IL-4,IL-2 and IFN-γ were extremely significantly improved (P<0.01).【Conclusion】 In this study,two synthetic peptide vaccines were successfully developed.P72-2 was higher than P72-1 in immune efficacy,and both could produce high level of specific antibodies,stimulate the proliferation of lymphocytes,induce the production of cytokines IL-4,IL-2 and IFN-γ.This study laid a technical foundation for the development of African swine fever vaccine.

【Objective】 This study was aimed to isolate exosomes from bovine mammary epithelial cell (BMEC) infected with Staphylococcus aureus (S.aureus),and investigate the effects of MOI(S.aureus:BMEC) and time post infection on the total concentration of exosomes.Then,the exosomes induced by S.aureus were also isolated and identified.【Method】 BMEC were infected with two levels of S.aureus (MOI=1 and MOI=10) for 3 h,and further cultured for 9,12,and 24 h after the infection.While,a blank control group with no infection of S.aureus was also set up simultaneously.The damage of the ultrastructure of BMEC caused by S.aureus infection was observed by scanning electron microscope.The exosomes in the supernatant of S.aureus-infected BMEC were then isolated by ultracentrifugation.The morphology,particle size and marker proteins of the exosomes were analyzed by transmission electron microscopy,nanoparticle tracking analysis,and Western blotting,respectively.The total amount of exosomes at different MOI and post-infection time was evaluated by detecting the total protein concentration of exosomes.Consequently,the optimal conditions for S.aureus in inducing the release of exosomes from BMEC were determined.【Result】 Scanning electron microscope observation showed that S.aureus infection induced shedding of microvilli,and destruction of cytoskeleton of the BMEC.Exosomes were successfully isolated and purified from the supernatant of S.aureus-infected BMEC by ultracentrifugation.Transmission electron microscopy showed that the purified exosomes were spherical vesicles with a lipid bilayer membrane,and a diameter of 30-150 nm. The morphology of the exosomes in each group was uniform.The total protein concentration of exosomes induced by S.aureus.with MOI=10 was higher than that at MOI=1 at 9,12 and 24 h after the infection respectively, and it was the highest at 12 h after infection.Furthermore,nanoparticle tracking analysis detected the average diameter of exosomes induced by S.aureus was about 116 nm.Western blotting results showed that the exosome protein markers CD9,CD81,and TSG101 were positively expressed.【Conclusion】 The results indicated that the exosomes were successfully isolated in this study by inspecting their morphological and molecular biological characteristics.The releasing of the exosomes derived from BMEC could be induced by S.aureus.And the highest protein content in the exosomes derived from the BMEC was observed after treated by S.aureus for 3 h and cells cultured exosome-freely for 12 h with MOI=10.

【Objective】 The aim of this study was to analyze molecular genetics and prevalent characteristics of Duck Tembusu virus (DTMUV) in Guangxi,so as to understand and master the new epidemic characteristics of DTMUV Guangxi strain,and provide data support for opportunely adjustment and formulation of effective prevention and control measures.【Method】 The tissue samples of duck collected in farms of Guangxi during 2019-2021 were detected for DTMUV by Real-time quantitative RT-PCR,the complete genome sequences of DTMUV were acquired by amplification and sequencing from partial positive samples according to detection date and origin area of DTMUV,and the sequence similarity,predominant amino acid,phylogenetic,recombination detection and estimation rate of evolution were analyzed.【Result】 The genome length of 8 DTMUV strains in Guangxi was 10 992 bp.The similarity of nucleotide sequence of open reading frame (ORF), E and NS5 genes among 8 DTMUV strains in Guangxi were 97.9%-99.8%,97.0%-99.9% and 97.8%-99.9%, and the similarity of amino acid sequence were 99.1%-99.9%,99.0%-100% and 99.3%-99.9%,respectively.The similarity of nucleotide sequence of ORF, E and NS5 genes between 8 DTMUV strains in Guangxi and reference strains were 86.4%-99.3%,85.8%-99.5% and 87.1%-99.2%,and the similarity of amino acid sequence were 96.0%-99.8%,94.8%-100% and 97.5%-99.9%,respectively.The similarity of TMUV with waterfowl source was higher than that of other host sources.Compared with vaccine strain FX2010,there were 11 amino acid mutations in DTMUV Guangxi strains,with the amino acids at 43,150,153,326,403,464 and 487 sites mutation were unique for DTMUV in Guangxi.Phylogenetic tree was constructed on ORF indicated that 8 DTMUV strains acquired in this study were belonged to 2.1 subgroup,while GX2011 and GX2015 strains also from Guangxi were formed to 2.2 subgroup,suggesting that the evolutionary trend of DTMUV epidemic strains in Guangxi was divergence.The phylogenetic trees of E and NS5 genes were similar with that of ORF.Recombination analysis results indicated that GXBH01-2019,GXZS02-2020,ziYY150901 and HB2016 strains existed recombination signal.Estimation rate of evolution performed on E and NS5 genes of DTMUV was 1.31×10^-3and 1.30×10^-3 substitution/(sites·year),which suggested that two genes kept similar evolutionary steps.【Conclusion】 At present,the epidemic strains of DTMUV in Guangxi were closely related to predominant prevalence strains,the E protein had unique amino acid mutation,the evolutionary trend was inconsistent,and there were molecular characteristics such as recombination,which provided basic data for formulating effective prevention and control measures.

【Objective】 The purpose of this study was to analyze the Brucella secreted protein BspI by bioinformatics,construct prokaryotic expression vector,and obtain BspI protein and prepare its polyclonal antibody,so as to provide materials for the follow-up study of the biological function of BspI protein.【Method】 Bioinformatics analysis of the amino acid sequence of BspI protein was performed using online software,referring to the BspI gene sequence (GenBank accession No.:DK63_1233) of Brucella 16M strain,after PCR amplification,BspI gene was ligated into pMD19-T cloning vector and screened for positive clones.The target gene was ligated into pET-28a expression vector,transformed into Escherichia coli BL21(DE3) competent cells,induced protein expression by IPTG,and analyzed by SDS-PAGE.The protein was purified by nickel column affinity chromatography, the purified protein was mixed with Freund's adjuvant to immunize rabbits,and blood was collected to isolate serum for analysis of polyclonal antibody specificity and antibody titer by Western blotting and indirect ELISA,respectively.【Result】 BspI protein was unstable hydrophilic protein,presented a transmembrane structure with no signal peptide region,had 13 phosphorylation sites and 7 antigenic determinants,and the secondary structure of mainly contained alpha helix,extended chain,and random coil.The 675 bp of BspI target gene was successfully amplified by PCR,and the pET-28a-BspI expression vector was successfully constructed.SDS-PAGE and Western blotting analysis results showed that the 25.3 ku protein was successfully expressed with no obvious heterobands after purification,and the prepared polyclonal antibody was able to specifically bind to BspI protein.The indirect ELISA results represented the polyclonal antibody titer of BspI protein was 1:409 600.【Conclusion】 The prepared polyclonal antibody against BspI protein of rabbit origin could specifically recognize BspI protein,and the protein was relatively reactogenic,which provided a reference for further research on the role played by BspI protein in the endoparasitism of Brucella.

【Objective】 This study was aimed to verify whether the NP protein of Bovine parainfluenza virus type 3 (BPIV3) could enhance the immune effect of BPIV3 inactivated vaccine【Method】 The antigenicity of the protein encoded by NP gene was analyzed by bioinformatics softwares,and the antigenic region was screened.The truncated NP gene sequence of BPIV3 was amplified by PCR and connected to pET-32a(+) plasmid.Then the high-purity BPIV3 NP protein was obtained by E.coli prokaryotic expression system and Ni affinity chromatography.It was confirmed by Western blotting.BPIV3 was inactivated with 0.3% formaldehyde and mixed with Freund's adjuvant 1:1 to prepare inactivated vaccine.Eight New Zealand White rabbits were randomly divided into four groups with two rabbits in each group,including inactivated vaccine group,NP protein group,inactivated vaccine and NP protein mixed group and control group.Blood samples were collected before and every 7 days after immunization.The levels of specific antibodies and neutralizing antibodies in New Zealand White rabbits of the four groups were measured and compared by indirect ELISA and virus neutralization test.【Result】 DNAStar analysis showed that the average antigen index of amino acid region 193-368 of NP protein was 0.4-1.7,and the hydrophilic index was 0-1.5,which proved that this region had strong antigenicity and hydrophilicity.The NP gene was amplified by PCR and the recombinant expression vector was constructed.Gene sequencing showed that the recombinant expression vector was consistent with the expected results.The results of SDS-PAGE showed that NP protein was highly expressed with a molecular weight of 50 ku and expressed in the form of inclusion body.Western blotting showed that the expressed protein had strong reactivity.The results of ELISA showed that 28 days after immunization,the specific antibody titer of the control group was 0,and the specific antibody titers of inactivated vaccine group,NP protein group and inactivated vaccine and NP protein mixed group reached 1:2¹¹,1:2¹⁷ and 1:2¹⁸,respectively.The results of virus neutralization test showed that 28 days after immunization,the neutralizing antibody titer of the control group was 0,and the neutralizing antibody titers of inactivated vaccine group,NP protein group and inactivated vaccine and NP protein mixed group were 1:2^3.32,1:2^4.48 and 1:2^4.98,respectively.【Conclusion】 BPIV3 NP protein could enhance the immune effect of BPIV3 inactivated vaccine.Adding NP protein to the inactivated vaccine could be used as a new vaccination method of BPIV3 inactivated vaccine.

【Objective】 The E0 protein of Bovine viral diarrhea virus (BVDV) was expressed by E.coli,after purification,the E0 protein polyclonal antibody was prepared by immunizing mice,which was then used for BVDV detection.【Method】 The BVDV E0 gene was amplified by PCR and ligated into pET-28a(+) to construct recombinant expression vector pET28a-E0.The recombinant plasmid was transformed into E.coli BL21 competent cells.After IPTG induction and affinity chromatography purification,the expression of E0 protein was identified by SDS-PAGE and Western blotting.The purified E0 protein was immunized into mice to prepare BVDV E0 polyclonal antibodies,and the specificity and titer of the polyclonal antibodies were detected by Western blotting,cell immunofluorescence,ELISA and other tests,as well as the application in BVDV virus detection.【Result】 The prokaryotic expression vector pET28a-E0 was successfully constructed,and the E0 protein was expressed and purified.The prepared BVDV E0 polyclonal antibody could specifically recognize the purified E0 protein and the total protein expressed by pET28a-E0 in BL21.Further,Western blotting,cellular immunofluorescence and double antibody sandwich ELISA proved that the prepared E0 polyclonal antibody could be used for the detection of BVDV.The results of indirect ELISA showed that the titer of E0 polyclonal antibody was higher than 1:64 000.【Conclusion】 The prepared BVDV E0 polyclonal antibody had a high titer and strong antigen-binding specificity,which provided material support for the biological functional study of BVDV E0 protein and the detection of BVDV.

【Objective】 This study was aimed to investigate the genome characteristics and variation of Porcine epidemic diarrhea virus (PEDV).【Method】 The small intestine tissues of 3 diarrhea piglets were used as templates,the pathogen was detected by RT-PCR.Using the RNA of the positive intestine tissue,RT-PCR technology was used to amplify the whole genome of PEDV. DNAStar software was used to edit and splice of the amplified sequence and perform similarity alignment.Mega X 10.0.5 software was used to construct phylogenetic trees of the whole genome and S gene of PEDV strain. RDP4 software was used to analyze the potential recombination events of PEDV strain.【Result】 PCR results showed that PEDV was detected in all the three sick piglets.The whole genome of PEDV strain was successfully obtained by RT-PCR fragmented amplification,named HB/HEBEU/2020.The virus genome size was 28 038 bp and it contained 7 open reading frames (ORF) which encoded replicase polyprotein 1a,replicase polyprotein 1b,spike (S),ORF3,envelope (E),membrane (M),and nucleoprotein (N) gene in 5' to 3' orientation.The similarity alignment of the complete genome as well as S gene between HB/HEBEU/2020 and PEDV variant strains,such as SNJ-P,USA/Colorado/2013 and HB2018 were 97.8%-99.3% and 95.7%-98.8%,respectively.Among all the commercially available vaccines,AJ1102 shared the highest similarity with HB/HEBEU/2020 strain,the nucleotide similarity between the complete genome as well as S gene of HB/HEBEU/2020 and AJ1102 were 98.3% and 97.3%,respectively.Genome-wide evolution results showed that 21 PEDV strains were classified into G1 genogroup and G2 genogroup,G1 genogroup was divided into G1a subgroup and G1b subgroup,G2 genogroup was divided into G2a subgroup and G2b subgroup,HB/HEBEU/2020 belonged to G2a subgroup.All the PEDV strains that belonged to G2 genogroup in the study were emerged after 2010.Among all the commercially available vaccines,AJ1102 had the nearest genetic relation with HB/HEBEU/2020,LW/L was second,the genetic distances between HB/HEBEU/2020 and CV777 as well as attenuated CV777 were far.3 possible recombination events were identified in the genome of HB/HEBEU/2020 strain.The recombinant regions of the three possible recombination events were 15 918-22 119,100-734 and 2 214-2 729 bp,respectively.The first two recombination events had high probability of occurrence.【Conclusion】 HB/HEBEU/2020 was a variant recombinant PEDV,which harbored distant phylogenetic relationships with classic PEDV strain CV777,but was closed related to the variant strains occurred in China after 2010.The results would provide theoretical and practical reference for postponing natural selection and evolution as well as formulating the vaccination procedure of PEDV.

【Objective】 The study was aimed to determine the etiology of diarrhea piglets in a pig farm in Jiangxi province.【Method】 The small intestine samples of piglets were tested for Porcine epidemic diarrhea virus (PEDV),porcine Transmissible gastroenteritis virus (TGEV) and Porcine rotavirus (PoRV) by RT-PCR.Positive sample was inoculated into MA104 cells for passage to isolate PoRV.The isolated strain was detected by electron microscope observation,indirect immunofluorescence test,PoRV VP4 and VP7 genes sequencing and animal regression test.【Result】 The results showed that porcine intestinal disease samples were incubated at 37 ℃ for 2 h with final concentration of 15 μg/mL trypsin and inoculated with MA104 cells,which could proliferate and passage on cells,and the 6th-generation began to show stable cytopathic effect. Electron microscopic observation showed that the virus particles were 61-70 nm in diameter,the average size was 65 nm,with short fibrils and smooth outer edges,similar to wheel-like particles,which had typical morphological characteristics of PoRV virus particles.Both indirect immunofluorescence assay and RT-PCR were positive for PoRV,and the isolated strain was determined to be PoRV.Sequence analysis of VP4 and VP7 genes revealed that VP4 genotype had the highest homology with P【23】 genotype, and VP7 genotype had the highest homology with G5 genotype,according to the latest classification method of group A Rotavirus,the isolate was classified as G5P【23】 genotype.The results of animal regression test showed that the one-day-old newborn piglets with oral infection of the isolate had watery diarrhea,vomiting and other clinical symptoms about 24 h after infection,and could detect PoRV in the feces.【Conclusion】 Through continuous passage of MA104 cells,a strain of PoRV was successfully isolated from the small intestine samples of diarrhea piglets in a pig farm in Jiangxi province,the isolated strain belonged to G5P【23】 genotype PoRV,which was the pathogen of diarrhea in piglets.

【Objective】 This study was aimed to prepare propolis inactivated vaccine of Riemerella anatipestifer (RA) epidemic strain in Guizhou.【Method】 The RA serotype 2 Guizhou epidemic strain (RA-SS-8 strain) was selected as the basic strain,and the bacterial growth curve was measured by spectrophotometry and plate counting method in turn,and then the inactivation conditions were screened.Propolis was used as an adjuvant to prepare RA Guizhou epidemic strain propolis inactivated vaccine,and conduct sterility test,safety test and immune duck challenge protection test were carried out.【Result】 Spectrophotometric determination of D_600nm value of strain RA-SS-8 with culture time showed that the bacteria proliferated slowly in the period of 0-3 h,and the proliferation trend was obviously accelerated in the period of 3-10 h,and the proliferation gradually became flat after 10 h,with the extension of the culture time,the bacteria finally entered the decay stage.The results of plate counting method showed that when the D_{600 nm} value of the RA-SS-8 strain was 0.1-0.8,the bacteria was in the logarithmic growth phase,and the D_{600 nm} value and the number of viable bacteria showed a good linear relationship. The best inactivation conditions of RA-SS-8 were 0.2% formaldehyde solution and 37 ℃ for 12 h.The bacterial content of the propolis inactivated vaccine was 3.8×10⁹ CFU/mL,and the dry matter content of propolis was 10 mg/mL.No colony growth was found on the chocolate agar medium in the sterility test.Ducks inoculated with 2 times of the immunization dose did not show adverse reactions during the observation period,and no obvious lesions were observed in gross lesions.The protection test of immunized ducks showed that the protection rate of ducks in the immunized group against RA-SS-8 strain was 70%,the propolis inactivated vaccine had good protective effect on the heart and liver tissues of the experimental ducks.【Conclusion】 The propolis inactivated vaccine of RA epidemic strain in Guizhou was successfully prepared,which laid a foundation for the preparation of propolis inactivated vaccine and animal immunity test.

【Objective】 The purpose of this study was to establish a method for expression and purification of large amount of porcine interferon-δ5 (pIFN-δ5),and analyze the effect of pIFN-δ5 against Porcine epidemic diarrhea virus (PEDV) infection.【Method】 According to the sequence of pIFN-δ5 in GenBank(accession No.:NM_001164854.1),primers were designed and PCR amplification was carried out with cDNA obtained from porcine liver tissue as a template.The target gene was ligated into the linearized pET-32a(+) vector digested with EcoRⅤ and Hind Ⅲ,the recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,the induction expression conditions were optimized,and detected by SDS-PAGE and Western blotting.pIFN-δ5 protein was abundantly expressed,purified using a nickel ion affinity chromatography column,and determined the protein purity.The interferon titer of pIFN-δ5 was detected by cytopathic inhibition method,the cytotoxicity of pIFN-δ5 was detected by CCK8 method,and the ability of against PEDV infection was further determined.【Result】 The recombinant expression plasmid pET-pIFNδ5 was successfully constructed.After exploring the induction conditions,it was found that when the D_{600 nm} value was 0.5 to 0.6,the IPTG concentration was 0.8 mmol/L,and the temperature was 37 ℃,the target protein pIFN-δ5 was mainly expressed in the supernatant of cell lysis.After massive expression and purification,pIFN-δ5 protein with purity of 95% could be obtained.The antiviral activity was determined by the VSV/MDCK titration system,and the specific activity was 5×10⁴ U/mg.CCK8 assays indicated that pIFN-δ5 had less effect on cell activity.Real-time quantitative PCR,Western blotting and indirect immunofluorescence results indicated that pIFN-δ5 exhibited significant resistance to PEDV infection.【Conclusion】 This study established a method for expression and purification of pIFN-δ5,and demonstrated that pIFN-δ5 had good activity against PEDV infection through a series of in vitro antiviral assays,which laid a foundation for the use of pIFN-δ5 as an antiviral agent and its clinical application.

【Objective】 The NS5A gene of Bovine viral diarrhea virus (BVDV) was expressed in vitro by prokaryotic expression system to obtain the non-structural protein NS5A.The nucleotide and amino acid sequences were analyzed to analyze the function of BVDV non-structural protein NS5A.【Method】 A pair of primers were designed and synthesized according to the NS5A gene sequence of BVDV-1 V006 strain(GenBank accession No.:KX170647) deposited in GenBank. The NS5A gene was amplified by PCR using the cDNA of BVDV GSTZ strain from yak as a template and was inserted into the pET-28a(+) vector,and the recombinant prokaryotic expression vector pET28a-NS5A was constructed. After preliminary identification by enzyme digestion and sequencing,E.coli BL21 (DE3) competent cells were transformed,and then induced by IPTG.The expression of the recombinant protein was identified by 10% SDS-PAGE electrophoresis and Western blotting analysis,and the genetic development and evolution tree was constructed according to the sequence of NS5A gene.The hydrophilicity,surface plasticity and antigenicity of NS5A protein were predicted by DNAStar software,and the B-cell antigen epitope of NS5A protein was predicted combined with the prediction of secondary structure.【Result】 The target gene fragment of NS5A amplified by PCR was 1 488 bp,which was consistent with the expectation.The results of double enzyme digestion and sequencing showed that the recombinant plasmid pET28a-NS5A was successfully constructed.The recombinant protein was identified by 10% SDS-PAGE electrophoresis and Western blotting,and the target protein with the size of 55 ku was expressed.The size was consistent with the expected results.Through the construction of genetic development evolutionary tree for different BVDV strain NS5A gene sequences,it showed that GSTZ strain NS5A belonged to BVDV-1 in genetic evolutionary characteristics. The hydrophilicity of NS5A protein were mainly located at amino acids 12-21, 32-69, 75-113, 120-135, 143-147, 152-163, 165-180, 215-230, 265-274, 296-340, 348-378, 389-447, 455-463, 469-495. The surface plasticity were mainly located at amino acids 14-18, 37-42, 76-81, 86-109, 154-160, 169-178, 218-228, 297-309, 348-358, 365-373, 414-442, 430-437, 454-460. And there were many flexible regions, mainly located at amino acids 14-21, 37-43, 67-82, 86-93, 97-110, 152-158, 169-179, 218-231, 240-255, 296-310, 313-328, 344-359, 364-373, 413-422 and 472-483. The B-cell antigen epitopes of NS5A protein were mainly located at amino acids 15-18,76-81,154-158,169-178,218-228,297-309,348-358,365-373 and 414-422.【Conclusion】 The non-structural protein NS5A of BVDV from yak was successfully expressed and identified.The phylogenetic tree showed that the BVDV GSTZ isolate was classified into BVDV-1.NS5A protein had good antigenicity.This study provided a basic plateform which provided reference for function and immunological characteristics of BVDV non-structural protein NS5A,and further study on the effect of NS5A protein on viral replication and relationship between biological function and protein structure.

Development of rapid,specific and sensitive detection method for emerging and re-emerging zoonosis and animal disease is critical for disease surveillance,prevention and control.CRISPR-Cas system,characterized by their sensitivity,specificity,and scalability upon DNA and RNA recognition,have been repurposed for the new generation of molecular diagnostic tools and are highly applied for pathogens detection.In the COVID-19 pandemic,CRISPR-Cas based SARS-CoV-2 detection kits (SHERLOCK and DETECTR) have been approved by USA,and CRISPR-Cas12 based SARS-CoV-2 detection kit developed in China has also been approved for marketing,implying that CRISPR-Cas assisted detection method could have a lot of uses in the future.This review firstly introduces the classification of CRISPR-Cas systems,especially CRISPR-Cas9/Cas12/Cas13,which are commonly used for pathogen detection. It also introduces the mechanism and the wider applications of CRISPR-Cas system. Meanwhile,the paper analyses the key technical issues in the development of CRISPR-Cas detection platform including sequence conservation of target genes,target enrichment and amplification,pathogens sensing with different signal readouts,and discusses the future development and application prospect of this method.

【Objective】 This study was aimed to explore the active ingredients and mechanism of action of Astragalus membranaceus (Fisch.) Bunge.on flora imbalance diarrhea in piglets based on network pharmacology and molecular docking.【Method】 The main chemical constituents and the potential targets of Astragalus membranaceus (Fisch.) Bunge.were obtained and collected by traditional Chinese medicine systematic pharmacology (TCMSP) database.Targets of flora imbalance diarrhea were derived from GeneCards and OMIM databases.The targets predicted by the two methods were mapped to obtain the common action targets. STRING database was used to construct the interaction relationship between target proteins,and Cytoscape_3.8.0 software was used to analyze the topology of the network.DAVID database was used to analyze the enrichment of co-acting targets.The core components and core targets were docked by AutoDuck_4.2.6 software.【Result】 There were 11 core components in Astragalus membranaceus (Fisch.) Bunge. which regulated flora imbalance diarrhea in piglets,corresponding to 65 target genes,854 disease targets and 25 intersection targets.Tumor necrosis factor(TNF),transcription factor AP-1(JUN),Caspase-3(CASP3),interleukin-6(IL6),NF-kappa-B inhibitor alpha (NFKBIA),IL1B,IL10,cellular tumor antigen p53(TP53),peroxisome proliferator-activated receptor gamma(PPARG) and C-C motif chemokine 2 (CCL2) were shown as key targets in PPI network.GO function and KEGG pathway enrichment analysis showed that the effective components of Astragalus membranaceus (Fisch.) Bunge.were involved in transcriptional regulation,immune response,cell response to lipopolysaccharide and other biological processes through American trypanosomiasis,inflammatory bowel disease,amoebiasis,cytokine-cytokine receptor interaction,influenza A,malaria,T cell receptor signal pathway and Salmonella infection.The results of molecular docking showed that the core components such as quercetin,kaempferol and formononetin were closely bound to the key targets such as TNF and IL1B,and had good affinity.【Conclusion】 The Astragalus membranaceus (Fisch.) Bunge.might have therapeutic effect on flora imbalance diarrhea in piglets by TNF,IL1B and other targets through main active ingredients such as quercetin,kaempferol and formononetin,and by participating in american trypanosomiasis,inflammatory bowel disease,malaria,T cell receptor signal pathway,Salmonella infection and other pathways.

【Objective】 This study was aimed to investigate the drug resistance phenotype and drug resistance gene carrying status of Acinetobacter pleuropneumoniae (APP),a pathogen of porcine contagious pleuropneumia (PCP) in some large-scale pig farms in Guangdong province,and to analyze and compare their correlation,so as to provide theoretical basis for effective prevention and control of PCP.【Method】 The dead pig materials of suspected PCP collected from large-scale pig farms in different regions of Guangdong province from 2019 to 2021 were isolated,identified and sequenced by pathogen isolation and culture,Gram staining,biochemical test,PCR amplification and other methods.Then the drug sensitivity test was carried out on the isolated strains,and their drug resistance genes were detected by PCR to determine their drug resistance phenotype and the carrying rate of drug resistance genes,and the consistency between them was analyzed and compared.【Result】 The isolates needed to be grown in culture plates containing serum and NAD.Gram staining results showed that the isolated bacteria were red,which could be determined as Gram-negative bacteria.Through biochemical test,PCR results and sequencing analysis,20 strains of APP were isolated,and did not show obvious regional characteristics.All strains showed multiple drug resistance,and about 50% of the strains showed 8 or more drug resistance.The drug resistance rates to tetracyclines,sulfonamides,chloramphenicols and macrolides were 77.5%,65.0%,55.0% and 48.8%,respectively.Further analysis showed that the isolated bacteria were mainly resistant to tetracycline,sulfaisoxazole,florfenicol and lincomycin,but sensitive to cefazolin (Pioneer V) and azithromycin.bla_CMY, aph(2″)-Ⅰb,sul1,sul2,sul3,tetA,tetB,tetM,tetO,tetR and floR were detected in 23 main drug resistance genes,and the carrying rates were 85%,85%,50%,60%,75%,30%,100%,85%,100%,70% and 80%,respectively.No quinolones,macrolides and lincomycin related resistance genes were detected.【Conclusion】 APP isolated from pigs in Guangdong province showed extensive drug resistance and multiple drug resistance.The drug resistance gene was basically consistent with the drug resistance phenotype,indicating that the carrying of drug resistance gene was one of the main reasons for bacterial resistance to antibiotics,but there might also be other undetected drug resistance genes or new drug resistance mechanisms.

【Objective】 The purpose of this experiment was to study the protective effect of traditional Chinese medicine (TCM) combined with probiotics on E.coli diarrhea in mice.【Method】 The best factor ratio of TCM combined with probiotics was selected by orthogonal test. 60 mice were randomly divided into blank group, model group, TCM group, probiotics group and TCM combined probiotics group, with 12 mice in each group. Mice in blank group were intraperitoneally injected with normal saline (0.2 mL/mouse), model group, TCM group, probiotics group and TCM combined probiotics group were intraperitoneally injected with E.coli isolate 1×10⁷ CFU/kg. The TCM group was gavaged with mixed TCM solution for 10 mL/kg(the concentration of dandelion extract was 0.25 g/mL, and the concentration of total flavonoids of Astragalus membranaceus and Astragalus polysaccharides were 0.05 g/mL), the probiotic group was gavaged with Bacillus subtilis suspension for 4 mL/kg (Bacillus subtilis contents 5×10⁷ CFU/mL), the TCM combined with probiotics group was gavaged with the mixture of TCM and Bacillus subtilis, they were given by gavage once a day for 3 days. The diarrhea, body weight and dietary quantity of the mice were recorded every day. 12 hours after the last administration, the heart, liver, kidney, spleen and lung were separated, weighed and the viscera coefficient were calculated. Hematological indicators were analyzed by blood test. Fecal and small intestine contents were taken to observe fecal occult blood and count bacteria. The activity of MPO in serum and small intestine were detected by ELISA.【Result】 The best factor ratio of TCM combined with probiotics was A₁B₂C₂D₂. The results showed that compared with blank group, the diarrhea rate of the model group was 100% without self-healing and there were obvious symptoms and pathological changes. The diarrhea index was extremely significantly increased (P<0.01), body weight and dietary amount were extremely significantly decreased (P<0.01), liver and spleen indexes were increased (P<0.05), the lymphocyte ratio and intermediate cell ratio were decreased (P<0.05) and the granulocyte ratio were extremely significantly increased (P<0.01) in model group. Compared with model group, diarrhea index in TCM group and TCM combined with probiotics group was extremely significantly decreased (P<0.01), body weight and dietary quantity were increased (P<0.05), liver and spleen indexes in TCM group were decreased (P<0.05), viscera indexes in TCM combined with probiotics group were extremely significantly decreased (P<0.01). LYM was increased significantly (P<0.05) and GRAN was decreased significantly (P<0.05) in TCM group, probiotics group and TCM combined probiotics group. Hematological indexes were greatly improved (P<0.05). Fecal occult blood test results showed that model group was strong positive, TCM group and probiotics group were weak positive, while TCM combined with probiotics group was negative. Compared with blank group, the number of E.coli in intestine in model group was extremely significantly increased (P<0.01), and the activity of MPO was extremely significantly increased (P<0.01). Compared with model group, the number of E.coli in intestine and the activity of MPO in serum and small intestine of mice in TCM group and TCM combined probiotics group were significantly decreased (P<0.05).【Conclusion】 TCM combined with probiotics can reduce diarrhea index, liver and spleen indexes, increase body weight and diet, turn fecal occult blood into negative, and reduce the number of E.coli in intestine and the activity of MPO in serum and small intestine, so as to provide a synergistic protective effect on E.coli induced diarrhea in mice.

【Objective】 This study was aimed to investigate the effect of Pseudorabies virus (PRV) infection on endoplasmic reticulum stress (ER) and unfolded protein response (UPR) in BHK-21 suspension-cultured cells.【Method】 The suspension-cultured baby hamster kidney cells (BHK-21) were infected by PRV at a multiplicity of infection (MOI) of 0.01.Samples were taken at 12,24,36,48 and 56 h after inoculation,respectively.Cell survival rate was detected by CCK-8 method,and virus titer was detected by Reed-Muench method,and the appropriate time of virus inoculation was screened.The uninfected cells were used as control group.Samples were taken at 12,24,36,and 48 h after infection,and the gene expression changes in the pathways related to endoplasmic reticulum stress marker GRP78 and UPR receptor proteins (PERK,IRE1 and ATF6) were detected by Real-time quantitative PCR,the expression of related proteins were detected by Western blotting.Endoplasmic reticulum stress inducer toxocarotene (Tg) of 0,0.001,0.005,0.01 and 0.02 μmol/L and endoplasmic reticulum stress inhibitor taurodeoxycholic acid (TUDCA) of 0,20,40,80 and 160 μmol/L were added to PRV at the same time.Cells were collected at 48 h to determine virus titer and cell survival rate.【Result】 The relative cell viability was less than 70% after 56 h,and the viral titer reached 8.1 lg TCID₅₀/mL at 48 h after PRV infection.Therefore,samples within 48 h (12,24,36 and 48 h) post PRV infection were selected to perform the analysis of endoplasmic reticulum stress.Compared with control group,the transcript levels of GRP78 were extremely significantly increased at 36 and 48 h by PRV infection (P<0.01).In the PERK pathway,the transcript level of ATF4 was extremely significantly increased at 36 and 48 h (P<0.01),and the transcript level of GADD34 was significantly increased at 36 h (P<0.05) and extremely significantly increased at 48 h (P<0.01).The level of eIF2α phosphorylation was extremely significantly increased at 36 and 48 h by PRV infection (P<0.01).In the IRE1 pathway,sXBP1 (spliced XBP1) was founded from 36 h after PRV infection.The transcript level of p58^IPK was significantly increased at 36 h (P<0.05),and the transcript levels of p58^IPK and EDEM were extremely significantly increased at 48 h(P<0.01).In the ATF6 pathway,there were no significant changes in the transcript levels of ERp57,PDI,Calnexin,and Calreticulin (P>0.05).Compared with 0 μmol/L group,cell viability were extremely significantly decreased by 0.01 and 0.02 μmol/L Tg,PRV titers were extremely significantly increased by 0.005 and 0.01 μmol/L Tg(P<0.01).【Conclusion】 Endoplasmic reticulum stress was induced,and PERK and IRE1 signaling pathways of UPR were activated by PRV infection in suspension-cultured BHK-21 cells.The PRV deployed endoplasmic reticulum stress to enhance its replication.

【Objective】 This study was aimed to understand the virulence and drug resistance of Haemophilus parasuis (Hps) serotype 7 epidemic strains in Henan province.【Method】 Taking the reference strain of Hps serotype 7 as the control,the 16S rRNA gene amplification,sequence analysis,virulence gene detection,pathogenicity test and drug sensitivity test were carried out on 6 strains of Hps serotype 7 isolated and identified from clinical cases.【Result】 16S rRNA gene sequencing and similarity comparison showed that the similarity between strain 1436 and standard strain 0007 was 100%,and that between strain 1565 and 1624 and standard strain were 98.6%. Phylogenetic tree analysis showed that 1436 had the closest genetic relationship with standard strain 0007,1624 was the farthest related to standard strain 0007.Six clinical strains and standard strain showed two virulence genotypes.All strains carried vta1,vta2, vta3,wza,nanH,cdtA,cdtB,cdtC and espP2 virulence genes,and five clinical strains also carried ompP2 virulence gene.In the guinea pig pathogenicity test,the clinical strain showed different degrees of virulence enhancement compared with the reference strain.The reference strain could not infect guinea pigs and did not show any clinical symptoms.The clinical isolates could infect guinea pigs,showing some clinical symptoms,the incidence rates were 20% to 40%,and the mortality rate was 0.The standard strain and clinical strains were sensitive to ceftazidime and ceftiofur,they were mediated to doxycycline and florfenicolo,the resistance to antibiotics such as neomycin,kanamycin,amikacin,erythromycin and tilmicosin was strong,and all strains showed multiple drug resistance.【Conclusion】 Hps serotype 7 clinical strains had certain pathogenicity,but their virulence was weak,they were sensitive to cephalosporins and had obvious multi drug resistance.Clinically,the prevention and control of this serotype should be paid attention.This study laid a foundation for the epidemiology and pathogenesis of Hps serotype 7,and provided a reference basis for the clinical prevention and control of Hps serotype 7.

【Objective】 This study was aimed to detect the distribution and expression of melatonin (MT) specific membrane receptors MT1 and MT2 in different parts of sheep gastric tissues by traditional molecular biological methods to preliminarily explore and clarify the difference of melatonin concentration and action mode in these areas.【Method】 The methods of ELISA, Real-time PCR, immunohistochemistry and Western blotting were used to detect the distribution and expression of melatonin specific membrane receptors MT1 and MT2 in sheep saccus ruminis dorslis, saccus ruminis ventralis, reticulum, omasum and abomasum.【Result】 The results of ELISA showed that melatonin was contained in all gastric tissues of sheep, and the content of melatonin in abomasum was the highest, followed by omasum, and that in saccus ruminis dorslis, saccus ruminis ventralis and reticulum were lower. The results of Real-time PCR showed that the contents of MT1 and MT2 genes mRNA were the highest in the abomasum, followed by saccus ruminis ventralis, and lower in saccus ruminis dorslis and reticulum. Immunohistochemistry results showed that MT1 and MT2 proteins were distributed in all gastric tissues of sheep, mainly expressed in the mucosal layer of each gastric tissue, and the distribution of MT1 and MT2 proteins gradually increased from the bottom to the neck in the abomasum glands. The results of Western blotting showed that the expression of MT1 and MT2 proteins was the highest in reticulum, followed by omasum, and lower in saccus ruminis dorslis, saccus ruminis ventralis and abomasum.【Conclusion】 Melatonin was differentially expressed in various gastric tissues of sheep so as to play a variety of physiological functions. It might regulate the physiological process of rumination after chyme stimulation through the signal transduction system by combining with the specific receptors MT1 and MT2 in gastric tissues.

【Objective】 This study was aimed to screen broader-spectrum and effective antibacterial proteins so as to lay the foundation for analyzing antibacterial mechanism of Lactobacillus plantarum thoroughly and exploring new antibacterial agents.【Method】 Lactobacillus plantarum GX20200417-1 was chosen to be the object of this study.Oxford cup method was selected to determine the antimicrobial activity.Proteins were extracted from Lactobacillus plantarum GX20200417-1 by the means of centrifugation at low temperature and ultrafiltration and their antibacterial activities were measured.The extracellular proteins were treated with different temperatures,pH and protease to study the physicochemical properties of antibacterial proteins.Antimicrobial components from Lactobacillus plantarum metabolites were further analyzed by LC-MS/MS.【Result】 Lactobacillus plantarum GX20200417-1 had strong antibacterial effect on Salmonella,Staphylococcus aureus and Escherichia coli.The antimicrobial activity of fermentation was tested at different time which was the strongest at 24 h with the antimicrobial diameter.The proteins extracted from fermentation had the similar antimicrobial activity with the fermentation.Lactobacillus plantarum antibacterial protein exhibited a high stability towards the temperatures. Compared with control group, there was no significant change in antibacterial activity at 20-80 ℃ (P<0.05),the best antibacterial activity at pH 6.0-8.0 and sensitive to protease.By GC-MS/MS analysis,five kinds of proteins were obtained which were not only corrected with bacteriostasis,but also had much more credibility.They were pediocin PA-1,lysin,polyketide synthase,accessory protein and LysM peptidoglycan-binding domain-containing protein and the molecular weight were 5.348,7.348,8.348,6.348 and 19.662 ku respectively.Pediocin PA-1 and lysin achieve bacteriostatic effect mainly by destroying bacterial cell wall and cell membrane.LysM peptidoglycan-binding domain-containing protein could recognize peptidoglycans including N-acetylglucosamine (GlcNAc) residue and upregulate the expression of antibacterial peptides.Accessory protein participated in the synthesis of bacteriocin,polyketide synthase participated in the synthesis of antibiotics,both of which played an indirect bacteriostatic role by participating in the synthesis of antimicrobial substances.【Conclusion】 In this study,five antimicrobial proteins of Lactobacillus plantarum GX20200417-1 were successfully identified,which laid a foundation for protein separation and purification.Its speculated that Lactobacillus plantarum GX20200417-1 could play an antibacterial role by the coordination of antibacterial proteins in metabolites.