This study was amied to learn the toxic effects of ricin on human peripheral blood B lymphocytes（IM-9）and the expression of related genes.The extracted ricin of different concentrations were added to culture cells for 6 and 12 h to make sure the median inhibitory concentration,then culture cells at the median inhibitory concentration for 2,6,10 and 12 h,at each time point detect the expression of immune related genes CD40,IL-1β,TNF-α and apoptosis related genes Bcl-2,Bax,Caspase-3.The results showed that the inhibitory effect of ricin on IM-9 cells was increased with the increase of concentration and time.The expression of TNF-α and CD40 genes increased significantly in experimental group than control group at 6 h（P<0.05).10 and 12 h reached extremely significant level（P<0.01);There was no significance of IL-1β gene expression between experimental and control groups（P>0.05).About apoptosis gene,the Bax gene expression decreased extremely significantly in experimental group at 10 h（P<0.05）and decreased significantly at 12 h（P<0.05);Bcl-2 gene had reached significant levels of four time periods（P<0.05), and 2,12 h were extremely significant（P<0.01);The expression of Caspase-3 reduced at 6 h,the other time points increased significantly（P<0.05),at 2 h reached extremely significant level（P<0.01).It showed that the gene expression of IM-9 cells could be significantly affected by ricin,TNF-α and CD40 genes were two potential genes to evaluate the toxicity by IM-9 cells.

In this study,ASB17 gene cDNA full length in Shaziling pig was cloned by RACE and it was analyzed by bioinformatics,the ASB17 gene expression levels were detected in Shaziling pig D30 period of nine different tissues（testis,muscle,intestine,lung,heart,kidney,liver,spleen,fat）and eight different developmental stages（E90,D1,D30,D60,D90,D120,D150,D180）of testicular tissue by Real-time quantitative PCR.The results showed that the length of ASB17 gene cDNA was 1 135 bp,and the ORF region was 888 bp,which encoded 295 amino acids.Real-time quantitative PCR results showed that the D30 period's expression level of ASB17 gene mRNA was the highest in testis,followed by fat and spleen,while the expression of ASB17 gene mRNA in muscle and small intestine tissue were the lowest,there were significant difference in testis and muscle,small intestine,lung（P<0.05);In the testis,the expression of ASB17 gene mRNA was the highest in D120 and D150 periods,while slightly declined in D180,the differences were extremely significant beween E90,D1,D30,D60 and D90,D120,D150,D180（P<0.01);The differences were not significant beween D90 and D180（P>0.05),while there were extremely significant with other periods（P<0.01);The sexual maturity periods of D120,D150,D180 had no significant differences（P>0.05).The full length of ASB17 cDNA was cloned and the expression levels of ASB17 gene in different tissues and different developmental stages were detected in this study,which laid the foundation for further research on the function of ASB17 gene.

This reasearch intended to find out the link between intramuscular fat（IMF）and DNA methylation level of AOC3,PPARG1 and SOD3 genes,and to make clear how DNA methylation influcence the fat deposition process.We used 10 F2 pigs with the extreme IMF of the Yorkshire pig×Min pig resource population.Longissimus muscle tissues were collected to find out the connection between the methylation level and IMF by using BSP.The results showed that there were no significant methylation differences in all the three candidate genes between the IMF extreme groups,which suggested that the phenotypic differences in this population might not be controlled by the degree of the methylation level,further study was still needed.

To expose the cat recombinant allergen Fel d 1 protein on the outer surface of hepatitis B core antigen（HBcAg）virus-like particles（VLPs), the recombinant Fel d 1（rFel d 1）was created by linking the two genes chain 1 and chain 2 that composed the Fel d 1 protein. Then the rFel d 1 sequence was inserted into the HBcAg c/e1 loop area, replacing the amino acids between D78 and E83 in HBcAg c/e1 loop area. We successfully constructed the prokaryotic expression vector pET28a-HBcAg-rFel d 1 via gene codon optimization and synthesis. The recombinant plasmid pET28a-HBcAg-rFel d 1 was transformed into E.coli BL21(DE3）cells, then induced by IPTG, purified by Ni-NTA affinity chromatography and tested by SDS-PAGE, Western blotting and transmission electron microscopy（TEM). The fusion protein HBcAg-rFel d 1 was expressed successfully in E. coli expression system and the pure fusion protein HBcAg-rFel d 1 was purified by Ni-NTA affinity chromatography. Further, TEM confirmed the fusion protein HBcAg-rFel d 1 could assemble into VLPs. The fusion protein HBcAg-rFel d 1 could assemble into VLPs spontaneously, which laid a solid foundation for the research of the preventive and therapeutic vaccines for cat allergy.

In order to study and analyze L1 gene of bovine papillomavirus（BPV）in Guizhou province,the L1 gene of BPV-GZ01 strain was amplified,cloned and sequenced using bioinformatic softwares and methods,and the secondary structure,tertiary structure,B-cell preponderant epitope,conserved domains analysis, transmembrane domain and signal peptide of L1 gene were predicted.The results showed that the length of L1 gene was 1 494 bp,encoding 497 amino acids.The L1 gene of BPV-GZ01 strain shared an amino acid identities of 98.6%,99.4%,98.4%,94.4% and 91.3%,and a nucleotide identities of 99.1%,99.8%,99.4%,87.6% and 82.8% with those of BPV2,BPV2-SW01,BPV2-AKS01,BPV13 and BPV1 strains,respectively.The results of phylogenetic tree analysis indicated that there was a close relationship between BPV-GZ01 and BPV2-SW01 strains.The prediction of secondary structure of L1 protein indicated that the random coil,extended strand and alphahelix took a higher percentage.The L1 protein was supposed contain 6 potential antigen epitopes.And no transmembrane domains and no signal peptide were found.The tertiary structure of L1 protein was curved spiral structure.These results provided a theoretical basis for immunologic diagnosis and further research of nucleic acid vaccine of BPV.

The study was aimed to establish a rapid and sensitive diagnostic method for the prevention and control of peste des petits ruminants.In this study,a fragment of PPRV N gene was amplified and cloned into pMD19-T cloning vector.Real-time quantitative PCR assay was performed using SYBR premix Ex Taq.The standard curve was plotted and the specificity,sensitivity and reproducibility of the assay were assessed.The generated standard showed linearity over the entire range from 2.82×10⁰ to 2.82×10⁷ copies/μL with a linear correlation（R²）of 0.992.The specificity of the assay showed that other viruses failed to show an amplification signal.The coefficient of variation（CV）values for intra- and inter-assay variability were low,ranging from 0.27%~2.77% and 0.41%~3.39%,respectively.The lower detection limit,based on plasmid copy number,achieved was 2.82 copies/μL and was 1 000 times more sensitive than conventional PCR assay.cDNA of 12 samples were tested using this method,9 were positive,and 3 were negative.The samples were also tested using conventional PCR,7 were positive and 5 were negative,proving that the two-step SYBR Green Ⅰ based Real-time quantitative RT-PCR assay reported here were more sensitive than conventional PCR.The establishment of this detection method is of great significance to rapid and sensitive diagnosis of peste des petits ruminants and preventing the spread of peste des petits ruminants.

The Nsp9 gene of porcine reproductive and respiratory syndrome virus（PRRSV）coded the RNA polymerase,which played an important role in the virus replication.In order to determine the interaction between the Nsp9 gene and virulence,replication capacity and cell tropism,the Nsp9 gene sequence of PPRSV vaccine strain CH-1R was cloned,and ligated to a virulent strain XH-GD infectious clone after restriction enzyme digestion,and transformed the virus under the basis of the reverse genetics.The results showed that the virus was successfully saved.And the results of biological characteristics test showed that titers and growth rate of saved virus were obviously higher than that of parental strain rXH-GD,but were lower than vaccine strain CH-1R.While plaques of two strains were similar in size.The polymerase of vaccine strain CH-1R could help to improve the drop degree of the virus,and this test could lay the foundation for further study of the effect of Nsp9 gene on virus virulence.

To study a novel vaccine of Toxoplasma gondii,the recombinant plasmids that cell penetrating peptides VP22 gene fused with one enhanced green fluorescent protein（EGFP)(pcDNA-VP22-EGFP)and three antigens of T.gondii（pcDNA-VP22-SAG1,pcDNA-VP22-GRA4 and pcDNA-VP22-AMA1）were constructed,respectively.The recombinant plasmids were identified by PCR,restriction endonuclease digestion and DNA sequencing.The pcDNA-VP22-EGFP plasmid was transfected into COS7 cells,and the expression of VP22-EGFP in COS7 cells was confirmed by fluorescence microscope and RT-PCR.The results showed that all recombinant plasmid were constructed correctly.Green fluorescence was observed successfully under the fluorescence microscope in transfected cells after 72 h.The gene fragment of 1 449 bp was obtained by RT-PCR.The results indicated that the recombinant plasmids were constructed successfully.

Teats number is one of the most important reproductive traits,and closely related to the economic benefit in pig industry.In order to reveal the underlying genetics of left teats number,right teats number and total teats number traits,a genome-wide association study（GWAS）was performed.Samples of DNA were collected to genotyping for 22 Kele pigs using the Illumina Porcine SNP 60K Chip.The GWAS was performed using a mixed-effects model and linear regression approach.When a genome-wide threshold was determined using the Bonferroni method（P<2.06E-5),4 single nucleotide polymorphism（SNP）markers were potentially associated with left teats number,right teats number and total teat number.However,3 SNPs were significant associated and 18 SNPs were potentially associated in chromosomes level.304 Ensembl genes were retrieved around 1 cM of the associated SNPs.The candidate genes in Wnt and Fgf signaling pathway（BTRC,FGF5,FGF8,BMP3,RASGEF1B and HMGB3）might have effect on target traits.These results provided valuable information about the selective breeding for Kele pigs.

To understand the Salmonella epidemic characteristics and pulsed field gel electrophoresis（PFGE）genotyping of goose,Anserindicus and Cygnus,33 strains of Salmonella were serotype typed by PFGE and genotyped with BioNumerics 6.6 cluster analysis software.The results showed that there were 16 PFGE patterns,which displayed 6 strains Salmonella paratyphi C,19 strains Salmonella thompson,1 strains Salmonella paratyphi B,1 strain Salmonella irumu,1 strain Salmonella oslo,5 undifferentiated.PFGE similarity is about 53.7% to 100.0%,and most of the strains and anthropogenic strains similarity was more than 80%.The results showed that goose,Anserindicus and Cygnus Salmonella serotypes same PFGE type with a high degree of similarity,and possibly from the same clone,quite different serotypes PFGE band pattern difference.

To predict the secondary structures and B-and T-cell epitopes of σB and σC proteins of avian reovirus（ARV),the secondary structures of ARV σB and σC proteins were predicted by DNAStar software and the Garnier-Robson,Chou-Fasman methods.B-and T-cell cell epitopes of σB and σC proteins were predicted by mutable parameters,including hydrophilicity,flexibility,surface probability and antigenicity.The analysis results showed that σB and σC proteins had few α-helix regions,meanwhile,compared with σB protein,σC protein possessed rather much β-sheet and turn regions,indicating σC protein exhibited more complex secondary structure.Furthermore,both σB and σC proteins had a few promising B cell and T cell epitopes,which would contribute to further explore the immune functions of σB and σC proteins,and provided a basis for development of ARV new type vaccine and therapeutic drugs.

To explore the feasibility of entering clinical trials of the goat Chlamydophila abortus eukaryotic plasmids,Chlamydophila abortus OmpA gene was amplified by PCR and cloned into the eukaryotic expressing plasmid pcDNA3.1(+）to construction the recombinant vetor.After identification by PCR and restriction enzyme digestion,this vector was transfected into PK-15 cells and its expression were observed by fluorescent antibody test,the distribution of serum antibodies and plasmid were detected in mice after the immunization of the recombinant vector and molecular adjuvant which was single-stranded DNA of E.coli bacterial genome.The results showed that the recombinant plasmid pcDNA3.1-OmpA was successfully constructed after detecting by PCR,enzyme digestion and sequencing.The OmpA gene was effectively expressed in PK-15 cells.The anti-OmpA antibody levels of the pcDNA3.1-OmpA with molecular adjuvant group was significantly higher than that in pcDNA3.1-OmpA group and the inactivated vaccine group at 14 d after immunization（P<0.05).This levels showed an upward trend following numbers of immunization and times.The highest levels was at 35 d after immunization.Then the antibody titers were gradually decreased which still maintain a higher antibody levels than before.The pcDNA3.1-OmpA could be detected in the heart,liver,spleen,kidney,lung,brain,jejunum and leg muscle of mice on 21 d after immunization,and couldn't be detected in any organs at 49 d after immunization.The results above indicated that the the recombinant vetor based on OmpA gene of Chlamydophila abortus was successfully constructed in this experiment,after joining the molecular adjuvant could induce to a higher level of serum antibodies in mice.

In order to study the transcriptional level of porcine PD-1 and its ligands in the disease state,and establish a Real-time PCR method for detection of porcine PD-1 and its ligands, three pairs of specific primers were designed according to porcine gene sequences of PD-1,PD-L1 and PD-L2,respectively.Fragments of target genes were amplified by PCR.The target genes were cloned into the multiple cloning site of pMD18-T vector.After being transfected into DH5α,recombinant plasmids were identified by PCR and genetic sequencing,as being standard samples for Real-time PCR standard curve.Specificity and repeatability were conducted.The results showed that it had a good linear inverse relationship between the Real-time PCR values of Ct and logarithm of template concentration.R² were all more than 0.99.Melt curves were the narrow single peak.The amplification products of Real-time PCR were the single band and no primer dimers.The coefficients of variation were less than 3% within and between groups of repeated test.It showed good specificity and repeatability.The mRNA levels of PD-L1（P<0.01）and PD-L2（P<0.05）were remarkably increased in the PBMCs of diseased pigs compared to healthy pigs,whereas no change was observed for PD-1(P>0.05).In this study,the methods of Real-time PCR for porcine PD-1,PD-L2 and PD-L1 mRNA were established,which laid a foundation for the study of the expression pattern of PD-1,PD-L1 and PD-L2 genes in pigs.

To establish a sensitive,specific and efficient method of antibody detection for porcine reproductive and respiratory syndrome virus（PRRSV),PRRSV N protein was expressed through prokaryotic expression system and purified by affinity chromatography to act as a coating antigen.Then an indirect ELISA detection method for serum PRRSV antibody was finally set up after the optimization of reaction conditions.Besides,the research also involved cross reaction,repeated experiments,and comparison with other ELISA kits.It was determined that the optimum concentration of coating antigen was 2 μg/mL and that the dilution ratio for serum was 1:100,with 30 min of incubation and 10 min of chromogenic reaction.With this method,positive serum samples of five common swine pathogens,including classical swine fever virus（CSFV),porcine circovirus（PCV）and porcine pseudorabies virus（PRV）and so on,were tested,and the results were negative.Both intra-assay and inter-assay coefficients of variation were below 10%,and the comparison with commercial ELISA kits indicated that its accuracy was 94.7%.So this indirect ELISA,which had been established in this research,could provide a rapid diagnosis for swine infected by wild PRRSV and applied in epidemiological investigation,as a convenient and efficient serological antibody detection method.

In eukaryotes,the regulation of gene expression plays an important role in the growth and development of organisms which is a multistep process that involves the transcription,translation and turnover of messenger RNAs（mRNA）and proteins.Researches have showed that the 3'untranslated region（3'UTR）of mRNA is emerging as critically important in regulating gene expression at post transcriptional levels.mRNA stability,localization and translation are largely determined by sequences in the 3'UTR.The aberrant regulation of 3'UTR will result in aberrant levels of expressed protein, enhancing metabolic changes and leading to disease.The researches of 3'UTR in the regulation of gene expression,human diseases and animal production were summarized and discussed in this paper to provide the reference for applications of 3'UTR in human disease diagnosis and treatment and animal performance.

The rumen degradations of TMR were studied in this experiment,in which cassava stems and leaves were used as roughage and formulated with concentrate in different proportions.The purpose of this study was to formulate the available TMR for finishing goat,and provide a theoretical basis for the applications of cassava stems and leaves in goat production.Six Hainan Black goats with permanent rumen fistulas were selected,the rumen degradabilities of DM,CP,NDF and ADF of TMR with 6 different concentrate to forage ratio（2:8,3:7,4:6,5:5,6:4 and 7:3）were tested using nylon bag method.The results showed that,the DM effective degradabilities at the ratio of 4:6,5:5,6:4,7:3 were significantly higher than that of 2:8 and 3:7（P<0.05).CP effective degradabilities at the ratio of 2:8,3:7,4:6 were significantly higher than that of 5:5,6:4 and 7:3（P<0.05).The NDF effective degradation at the ratio of 5:5 was significantly higher than that in the other groups（P<0.05).The ADF degradation at the ratio of 3:7 was significantly higher than that of 4:6,5:5, 6:4 and 7:3（P<0.05).The results indicated that,the nutrient degradation in rumen were higher when the concentrate to forage（cassava stems and leaves）ratio in TMR were 3:7,4:6 and 5:5,and the recommended ratio range were 4:6 to 5:5 in production practice.

This experiment was conducted to study the effects of mannan oligosaccharides with different adding schemes on growth performance, fecal microorganism and serum immune indexes of calves.Twenty newborn Holstein calves were randomly divided into 4 groups with 5 replicates per group.The claves in group Ⅰ（control group）were not fed the mannan oligosaccharide.The calves in group Ⅱ were fed 5 g mannan oligosaccharide added into the milk,and that of group Ⅲ were fed 5 g mannan oligosaccharide added into the starter diet while calves of group Ⅳ were fed 2.5 g mannan oligosaccharide added into the milk and the starter diet,respectively.The feeding trial lasted for 56 days.The results showed as follows:①During the whole experiment period,body length an body structure of calves had no significant difference between the groups（P>0.05).②During the whole experiment period,the average daily dry matter intake（ADMI）and average daily gain（ADG）in the experiment groups was higher than the control group,the F/G was lower than the control group,and group Ⅱ was the best of the three experiment groups.③On 21th day,the number of Bifidobacterium in feces of the calves in the group Ⅰ was significantly lower than that in other groups（P<0.05),and that of group Ⅱ was the highest;The number of E.coli in feces of the calves in the group Ⅰ was significantly higher than that in other groups（P<0.05),and the group Ⅱ was the lowest.On 56th day,the number of Bifidobacterium of experimental groups were higher than that of control group.The change of E.coli number of four groups were as the same as 21th day.④On 21th day,the serum IgA content of experimental groups were higher than group Ⅰ,and that of group Ⅱ were significantly increased（P<0.05);IgG had a similar change with the IgA;The serum IgM content of group Ⅳ was significantly higher than group Ⅰ（P<0.05), while that of group Ⅲ was lower than control group;On 56th day,the serum immune indexes of experimental groups were all higher than control group（P>0.05).In conclusion,adding mannan oligosaccharides could promote calf growth performance,gut health and immunity,while adding mannan oligosaccharide to milk had better effects.

This study was conducted to compare the effect of active dry yeast（ADY)and yeast culture（YC）on gas production,dynamics of gas production,methane content and rumen fermentation parameters by in vitro gas production technique,and the values of ME,OMD and yield of MP were also predicted according to 24 h gas production,in order to further reveal the mechanism of the difference effect and lay a foundation for those reasonable application.The results showed that ADY and YC had similiar effect on gas production, however,the gas production of YC group at 12,24 and 48 h were higher than that of ADY;Compared to CON group,the rate of gas production in earlier fermentation period was increased by 19.4% in YC group and decreased by 15.3% in ADY group（P>0.05),and that of YC group was significantly increased by 41.0% than that of ADY group（P<0.05);The content of CH₄ of YC group was significantly increased and content of H₂ and CO₂ were significantly decreased（P<0.05).The content of H₂ in ADY group was significantly decreased（P<0.05),while the content of CH₄ and CO₂ were not significantly changed（P>0.05);The pH and total VFA of rumen fermentation fluid in ADY and YC groups were not significantly changed（P>0.05),while the concentration of NH₃-N of the two groups was significantly increased（P<0.05).ADY had no significant effect on individual VFA（P>0.05),whereas YC tended to increase the ratio of acetic acid and decrease the ratio of isobutyric acid;Comparing to CON group,the OMD,ME and MP of ADY and YC groups were not significantly affected（P>0.05), while that of YC group were all significantly increased than ADY group（P<0.05).In conclusion,there were certain differences in the effect of ADY and YC on gas production,concentrate of gas,the rate of gas production and parameter of rumen fermentation.

In modern pig production,early weaning is a bottleneck problem which are restricting the development of pig industry.It can lead to early weaning syndrome in piglet,such as low appetite,growth inhibition,diarrhea and so on.One of the reasons induced these symptoms is impairment of intestinal mucosal barrier in piglets.The intestinal microflorais is one of the important parts of intestinal mucosal barrier.Therefore,study on the intestinal microflora of weaning piglets and nutritional regulations have been attracted more and more attentions,and it has an important influence on avoiding diarrhea,promoting the healthy growth and improving growth performance.The change of intestinal microflora in weaning piglets and research development of some common feed additives（microecologics,plant polysaccharide,acidulant,microelement,Chinese herbal medicine and plant extracts）for intestinal microflora were reviewed in this paper.

The objective of the study were to investigate the effects of low protein diets supplemented with amino acids on the reproductive performance,change of backfat thickness and milk composition of the lactating sows.35 crossbred sows with similar parity,body weight and body conditions were selected and randomly divided into 3 treatments and each treatment had 13,11 and 11 sows,respectively.A single-factor experimental design was used.Pigs in control group were fed conventional commercial diet with CP level of 17.7%,and the pigs in experimental groupⅠand Ⅱ were fed the diets supplemented with amino acids,and their CP level were 15.7% and 13.7%.The amino acid concentrations were the same in all three diets.The lactation length was 21 days.The results showed that:①Compared with control group,the total litter weight at 21 days,feed intake of lactating sows,survival rate and feed intake of piglets in the experimental groups had no significant difference（P>0.05).With the decrease of protein level,sows' body weight loss increased,but the differences were not significant among the treatments（P>0.05).②With the decrease of protein level,the ratio of sows' reduced fat thickness declined,but the differences among the treatments were not significant(P>0.05).③The milk composition of sows in experimental group Ⅱ was better than that in the control group,but the difference was not significant（P>0.05).The results indicated that using the diets with reduced CP level（from 17.7% to 15.7% or 13.7%）and supplemented with amino acids did not affect the performance of sows and piglets,and it would be an effective measure saving protein resource.

This trial was conducted to study the effect of Monascus compound additives on growth performance and meat quality of broilers.The total of 400 one-day-old Avian broilers were randomly allotted into 4 groups with 5 replicates per group and 20 birds per replicate.The broilers in group Ⅰ(control group),were fed a basal diet,and those in experimental groups Ⅱ,Ⅲ and Ⅳ were fed the basal diet supplemented with 0.5%,1% and 1.5% Monascus compound additives,respectively.The experiment lasted for 42 d.At the ending of the experiment,all the birds were weighted and two of each replicate were slaughtered,the samples from breast and leg muscles were taken for meat quality measurement.The results showed as follows:Compared with control group,the ADG increased significantly（P<0.05),and F/D decreased significantly（P<0.05）in group Ⅲ and Ⅳ,but the differences were not significant between group Ⅲ and Ⅳ(P>0.05);Compared with the control group,the muscle redness（a^*）and yellowness（b^*）in all the experimental groups increased significantly（P<0.05),and the drip loss rate decreased significantly（P<0.05),but the muscle lightness（L^*),cooking loss rate and shear force had no significant change（P>0.05),and pH_{24 h} of muscles in groups Ⅲ and Ⅳ reduced significantly（P<0.05).In conclusion,adding Monascus compound additives in diets could improve growth performance and meat quality in broiles, and the appropriate level should be 1%.

To mimic muscle development of Duolang sheep in vitro,we employed a two-step digestion method to separate satellite cells（SCs）and a differential adhesion method to purify the cells in Duolang sheep.Moreover,observation of microscopic images and immunofluorescence were used for identifying Duolang sheep SCs and its myogenic differentiation.Using immunofluorescence for Desmin,Pax7 and MyoD1 genes,we demonstrated that these marker genes all expressed in the SCs.The SCs formed significant myotubes when the serum was withdrawal from growth media,confirmed by the immunofluorescence for MHC and microscopic images.Taken together,we ssuccessfully isolated SCs and established the myogenic differentiation of SCs.

In order to compare the difference of milk fat globule membrane（MFGM）proteins between dairy cow and goat milk,30 milk samples were collected in a dairy farm and a goat farm from Anhui area,respectively.Extracted proteins from the MFGM-enriched fractions were identified and quantified by LC/MS approach.The results showed that 284 and 334 proteins of MFGM from dairy cow and goat were identified,the biological processes of MFGM proteins were similar between dairy cow and goat which were mainly related to biological regulation,localization,transport,signal transduction and response to stimulus.Meanwhile,there were some differences in molecular functions,and protein binding and nucleotide binding were the most prevalent molecular functions in dairy cow MFGM proteins,while protein binding and structural molecule activity were the most prevalent molecular functions in goat MFGM proteins.And structural molecule activity was the main molecular functions among the difference proteins.Pathway analysis revealed that tight junction,axon guidance,antigen processing and presentation,complement and coagulation cascades were enrichment in dairy cow MFGM proteins,and regulation of actin cytoskeleton was enrichment in goat MFGM proteins.Those results revealed the protein expression pattern difference between MFGM protein of dairy cow and goat milk,and provide reference data for further exploring the molecular mechanism of synthetic milk fat globule.

The growth hormone（GH）regulated diverse functions of the target cells through binding its receptor and played important roles in the process of metabolism of the body's growth and development.In order to obtain high purity chicken growth hormone（cGH）with biological activity,the gene segment of the mature cGH with six histidine at the C terminus was cloned into the expression vector pPIC9K.The recombinant plasmid（pPIC9K-cGH）was transformed to Pichia pastoris（GS115）by electroporation to construct the recombinant Pichia pastoris（GS115-pPIC9K-cGH).The GS115-pPIC9K-cGH secreted the recombinant protein（cGH）whose molecular weight was about 25 ku induced by 1% methanol induction for 96 h.The purity of cGH attained at least 95% through using Ni affinity chromatography and polyacrylamide glucan gel chromatography to purify the recombinant protein.The results of Real-time quantitative-PCR showed that the purified cGH recombinant proteins could up-regulate its target gene insulin-like growth factor Ⅰ（IGF-Ⅰ）mRNA in chicken liver cancer cell（LMH).It certified that the recombinant cGH protein had biological activity.This study laid a foundation for research of the structure,function and application of cGH.

The opportunistic pathogen Pseudomonas aeruginosa is a gram-negative bacteria which can form biofilms.In this review,we summarized the biology mechanism of biofilms in Pseudomonas aeruginosa,including Pseudomonas aeruginosa adhesion,extracellular polysaccharide Psl and Pel,alginate participated in the biofilm maturation,and quorum sensing systems regulated the formation of biofilm in Pseudomonas aeruginosa,and the drug target on biofilm.The bacterial biofilms protected the bacteria from unsuitable environmental conditions.Through researching the structure and the pathogenesis of Pseudomonas aeruginosa,and its resistance molecular mechanisms,we could adjust or regulate the expression of biofilm-associated factors,optimize the anti-infection treatment in Pseudomonas aeruginosa.

This study was aimed to analyze the association of four SNPs of MAP3K5 gene with growth and feed efficiency related traits in Duroc populations. Four SNPs(Ssc1:30769583 A>C;Ssc1:30781169 A>G;Ssc1:30940839 A>G;Ssc1:30962276 G>A)of MAP3K5 gene in Duroc populations were obtained by porcine SNP60 BeadChip（Illumina). The genotype frequency and allele frequency of four SNPs of MAP3K5 gene were analyzed,the result showed that four SNPs were the low-moderate mutation SNPs.The polymorphism of MAP3K5 gene and its association with feed efficiency related traits in Duroc population were analyzed. The results indicated that CC genotype individuals in Ssc1:30769583 A>C had significant lower RFI（133.08 g/d）than AA genotype individuals（P<0.05);GG genotype individuals in Ssc1:30781169 A>G had significant lower RFI（116.18 g/d）and ADFI（0.23 kg/d）than AG genotype individuals（P<0.05);GG genotype individuals in Ssc1:30940839 A>G had significant lower FCR（0.10%）than AG genotype individuals（P<0.05);AA genotype individual in Ssc1:30962276 G>A had significant lower ADG（0.04 kg/d）than GG genotype individuals（P<0.05). All in all,the results suggested that four SNPs polymorphisms of MAP3K5 gene had the significant impact on RFI, ADFI, FCR and ADG traits in Duroc pigs.

This study was aimed to investigate the genetic diversity of swamp buffalo breeds in China.The analysis of genetic diversity was performed in 40 buffalo individuals from 8 buffalo breeds（Dechang buffalo,Dehong buffalo,Wenzhou buffalo,Guizhou buffalo,Xilin buffalo,Fuzhong buffalo,Murrah buffalo and Nili-Ravi buffalo）by 30 microsatellite loci and LabChip chip test method.The results showed that 332 alleles at 30 microsatellite loci were found in 8 buffalo breeds,the average values of gene diversity and PIC were 0.7808 and 0.7554,respectively.Cluster analysis indicated that Dechang buffalo and Dehong buffalo firstly clustered together,followed by Fuzhong buffalo,Guizhou buffalo,Wenzhou buffalo and Xilin buffalo.Moreover,Murrah buffalo and Nili-Ravi buffalo clustered together.Our findings revealed that the 30 microsatellite loci could be used as an effective genetic marker for the analysis of genetic diversity among the buffalo breeds,which enriched the current SSR marker resources in buffalo.

In order to investigate the expression pattern of C-type natriuretic peptide（CNP）and its receptors（NPR）in buffalo follicles,firstly,the mRNA expression level of ANP,BNP,CNP,NPR1 and NPR2 in ovary were assayed by Real-time quantitative PCR;Secondly,the protein expression of CNP and NPR2 in buffalo follicles were detected by immunohistochemical stainning method;Lastly,the mRNA expression level of CNP and NPR2 in granule cells and cumulus cells were assayed by Real-time quantitative PCR.The results showed that CNP gene and its receptor NPR2 mainly expressed in buffalo ovary,the protein of CNP and NPR2 expressed in all stages of follicles,CNP mainly expressed in mural granulosa cells and NPR2 primarily presented in cumulus cells,the mRNA expression of CNP gene in granule cells was significantly higher than cumulus cells（P<0.05),whereas the mRNA expression of NPR2 gene in cumulus cells was significantly higher than granule cells（P<0.05).In conclusion,among the main members of natriuretic peptides and its receptors,CNP and NPR2 presented strong expression in buffalo ovary.CNP mainly expressed in mural granulosa cells,but NPR2 primarily presented in cumulus cells which arounding oocytes.

The study was mainly focused on the genetic characteristics of frizzled feather trait,the phenotype difference between frizzled feather（FF）and incomplete frizzled feather（IFF),and SNP analysis of the candidate gene KRT75 in the frizzle and incomplete frizzle chickens of different feather colors,which could provide scientific gist for the frizzle gene mapping,development and utilization of the two kinds of chicken.The resource population（homozygous Yellow feather Kirin chicken×Huaixiang chicken）of hybrid F2 was established.The back,neck,wing primary and wing-bow feather were observed and the scatter diagram of rachis bending degree was drawn.The blood of 10 white frizzle feather Kirin（WFF）chicken,10 Yellow frizzle feather Kirin（YFF）chicken,10 Royal,25 Yellow feather incomplete frizzle（YFIF）chicken and 15 Black feather incomplete frizzle（BFIF）chicken from homozygous WFF chicken×Royal chicken of hybrid F1 were extracted,then the DNA of those samples were extracted.The primer of KRT75 was designed,the PCR products were also sequenced after purification,Chromas 2.22 and DNAMAN software were used to analysis of sequence peaks photos and sequence alignment,respectively.The amount of orthogonal and reciprocals cross in hybrid F1 was 127 and 139, respectively, which all were the slight（incomplete）frizzled feather phenotype,with no different penetrance.The resource population of hybrid F2 were consisted of 55 frizzle chicks including some hens and cocks,106 incomplete frizzled and 68 contour feather chicken,the proportion of which was coincided with Mendelian segregation ratio of 1:2:1 by χ²-test（P>0.05),therefore it preliminary verified that the frizzle gene F was autosomal incomplete dominant inheritance.FF and IFF curved upward deviating from the skin,and the line slope of trend line of FF was 3.5 times than that of IFF.It found that there was no 69 bp deletion mutation in exon 5 region of KRT75 gene in WFF and YFF chicken,but 3 SNPs（T71C,T83C,C95T）were deleted in the 69 bp,which were homologous CC in WFF and YFF chicken,of two were heterozygous in YFIF chicken,while all were heterozygous in BFIF chicken.2 SNPs（T662C and T770C）were also deleted in exon 6,heterozygous of which were only deleted in BFIF chicken.The haplotype analysis indicated that 9 haplotypes were detected in 60 individuals,hap1/hap1 was specific genotype of WFF and YFF chicken.The haplotypes of two incomplete frizzle feather chicken were apparently different,hap4 was specific haplotype of YFIF chicken,while hap6,hap7,hap8 and hap9 were specific haplotypes of BFIF chicken.The frizzle gene F was autosomal incomplete dominant inheritance,there were obvious differences between FF and IFF traits,5 SNPs in exon 5 and 6 of KRT75 gene probably were the reference of molecular markers as distinguishing between frizzle and incomplete frizzle chicken.

In order to obtain the cloned sheep using bone marrow mesenchymal stem cells（BMSCs）as the donor cells,the BMSCs of sheep were chosen and reconstructed embryos were built to transfer.10 published microsatellite markers were chosen,and the DNA samples from clone sheep,donor cells and surrogate ewes were amplified,and the relationship of father-son（RCP）was analyzed using the Quantity One for genotyping.The results showed that the reconstructed embryos were successfully built for electric fusion using sheep BMSCs as nuclear donor,and making nuclear transplantation into enucleated mature oocytes of which the fusion rate was 80.62%.20 surrogate ewe were chosen to be implanted with the reconstructed embryos at morula stage by implant surgery,and 5 lambs were born and only 3 were survived.The genotype of cloned sheep was in line with the dornor cell and the RCP were more than 99.999%.In conclusion,the first clone sheep were obtained successfully by using BMSCs as a nuclear donor in this experiment.

This study was conducted to investigate the effect of PGI₂ on early embryo development of sheep in vitro lacking of endogenous prostaglandins（PGs).The specific inhibitor NS398 and SC560（specifically inhibiting the activity of COX-1 and/or COX-2）were added into the culture medium respectively to study the effect of COX-1 and COX-2 on embryonic development of sheep.Different concentrations of Iioprost were added respectively into the culture medium to investigate the effect of PGI₂ on early embryo development of sheep in vitro.The results showed that there were no significant differences on the cleavage and blastocyst rates between the group only with NS398 and that with both NS398 and SC560（P>0.05).There were significant differences between the group only with NS398 and control group（P<0.05).After adding NS398,different concentrations of exogenous Iloprost could replace the role of embryonic-derived PGI₂,which substantially eliminated the adverse effects of COX-2 inhibitor on early embryo development in vitro.The appropriate concentration of Iloprost was 1×10^-6 mol/L.H33342 staining showed no significant difference（P>0.05).The results demonstrated that embryonic endogenous COX-2 played a major role in regulating synthesis of PGI₂ in early embryo of sheep.The exogenous Iloprost was able to compensate for the loss of endogenous PGI₂ in embryos and remove the inhibition of NS398 on COX-2,ultimately promoting the development of early sheep embryos in vitro.

To analyse the genetic variation of porcine epidemic diarrhea virus（PEDV),one pair of primers was designed and RT-PCR was used to amplify the S1 gene epitope sequences of 4 PEDV field strains.Phylogenetic analysis showed that 4 strains were closely related to each other and belonged to the second group,and the Hunan and Henan isolates had a close relationship with JXGZ2013,GDZQ2012,GDZQ2014 strains,with the nucleotide homologies at 98.3% to 98.7%;Shanghai strain had a closed relationship with BJ-2011 strain and LZW isolates,with the higher homology at about 98.7%;Fujian isolates had a close relationship with strain of American in 2013,Japanese and Taiwan variation of China with the nucleotide homology at 99.0% to 99.5%;These results showed that a rapid variation and evolution of PEDV strains occurred in recent years,and linear B-cell epitope analysis showed that the threshold values at site 265 to 278 amino acid of 4 filed strains were higher than that of vaccine strains.The epitope differences indicated a possible of infectious that cause of patterns of epidemiology of PEDV.

Uterine disease after parturition is present in up to 40% of dairy cows and can cause cow infertility,which restricts efficiency of the cow production and leads to large economic loss.System administration and intrauterine infusion are widely utilized to treat cow endometritis.In recent years,the balloon technique is used gradually.Uterus washing pipe is used to inject liquid into the uterus and then the liquid and the inflammatory substances are discharged by the veterinarian.After the inflammatory substances cleaning up,the drug is delivered into the uterus.In order to provide a new method that can treat the infertility in dairy cows with metritis,323 cows which were diagnosed with metritis were used to detect the efficiency of balloon technique,the location of the balloon and the different drugs perfusion against infertility in dairy cows with metritis.The results showed that the balloon technique was showed remarkable effect against cow metritis comparing to the conventional technique.The pregnancy rate of the balloon technique was more than 60%,by contrast,the conventional technique was about 35%.Compared with being placed in the cornua uteri,the efficacy was more obvious when the balloon was placed in the uterine body,and the pregnancy rate was more than 60%.However,the pregnancy rate of another method was almost 40%.There were prominent curative effect of the three drug combinations.The pregnancy rates of the saline+Oxytetracycline,Lutajing+Gongdekang and saline were more than 70%,60% and 55%, respectively.Therefore,compared to the conventional technique and cornua uteri technique,balloon technique（uterine body）significantly increases the pregnancy rate,which is beneficial to be widely used in production.So it is significant that the new technique used widely is important for improving the production efficiency of dairy cows.

The study was aimed to prepare vaccines with different adjuvants,and research its effects on immunogenicity.The PCV2 Cap gene with its signal peptide removed was connected to pET-28a vector,and then was induced to express,using sodium deoxycholate（DOC）and low concentration of urea to dissolve the inclusion body.Different adjuvants,such as alum-based adjuvants,liposome adjuvants,propolis adjuvants,white oil adjuvants and Freund adjuvant were prepared,together with protein purified to make up subunit vaccines,and commercial inactivated vaccine as positive control,immuning mice,and ELISA method were used to detect changes in concentrations of animal serum antibody and cytokines,evaluated the immune protective effect.Results showed that the expression product in the form of inclusion body,using the DOC could omit dissolving inclusion body protein renaturation steps,and the purification method was simple,the capsid protein obtained had high purity.ELISA assays showed that PCV2-Cap had good immunogenicity.And we found that the water system adjuvants had high immune activity,this could provide important previous experimental data for the commercialization of the subunit vaccine.

In order to study the pathogenicity of the pathogenic bacteria in the vertical scale disease of Tilapia,one strain of pathogenic bacteria was isolated from different parts of the diseased Tilapia,named as GXKJDX01.Then the methods of 16S rDNA gene sequence analysis,bacterial morphology observation,physiological and biochemical identification,drug sensitivity test and artificial infection test were used.The results showed that the pathogenic strain GXKJDX01 was Aeromonas veronii,strain GXKJDX01 was not sensitive to 5 medicines,including penicillin G,erythromycin,vancomycin,and had a strong pathogenic effect in yellow-head catfish meat,Tilapia,loach and Procypris merus.11 kinds of drugs,including of enrofloxacin,levofloxacin,ceftriaxone,could be effective to the prevention and control.This study is the first report on the case of the pathogenic bacteria of the lepidorthosis,which was the Aeromonas veronii,and the theoretical basis was provided for the effective prevention and control.

To determine the pathogens that caused the death of the 10-day-old sheldrakes of a outbroke epidemic duck farm at the end of June 2015,at Cangshan District in Fuzhou,the liver,spleen and brain of dead sheldrakes were collected,and then tested by virus isolation,isolation and purification of bacteria,PCR detection,serology and drug susceptibility test.The results showed duck hepatitis virus type 1 and 11 type Riemerella anatipestifer were detected and isolated,and the isolated duck hepatitis virus type 1 had the highest homology with MPZJ1206 strain and GD strain,and the percentage of homology were 98.7%.Laboratory isolation and identification of pathogen determined that the case was duck hepatitis virus type 1 and serotype 11 Riemerella anatipestifer mixed infection.

In order to study the effects of storage conditions on survival and migration of three kinds of Salmonella,S.enteritidis,S.typhimurium and S.pullorum were artificially contaminated on eggshell.Different environment temperatures（4,25 and 37℃）and relative humidity（50% and 90%）were seted,and the number of Salmonellas on the surface of eggshell and egg contents were counted and detected on various time points.The results showed that 4℃ obviously reduced the number of three kinds of Salmonellas.At 50% RH,the number of three kinds of Salmonellas were reduced on eggshell surface at 25 and 37℃ environment.At 90% RH,despite the individual time points,the number of S.enteritidis and S.typhimurium presented downward trend at 25℃.And at the 37℃,the number of S.pullorum rapidly reduced,and the number of S.enteritidis and S.typhimurium firstly decreased and then increased,and finally maintained at the level of inoculated magnitude.The results of egg content detection showed that S.enteritidis and S.typhimurium were more likely to be detected in high temperature and high humidity environment,while S.pullorum was not detected in all kinds of experimental environment.The results suggested that low temperature and low humidity reduced Salmonellas survival on eggshell and inhibited it migration into the egg,and high temperature and high humidity could promote Salmonellas proliferation,increase the risk of its penetration into egg.There were some differences in the survival on eggshell and invasion between the different serotypes Salmonellas.

This test was aimed to research the role of rafoxanide on anthelmintic effects in six months old Yili horses.Ten Yili male horses with same date of birth,body weight（117.60±15.84)kg and the horses of six months old were randomly divided into 2 groups,each group of 5 horses,control and trial groups,respectively.The horses in two groups were fed with the same nutritional levels of roughage and concentrate supplement.Horses of trial group were expelled passasite by rafoxanide,but control group had not treatment.After expelling passasite,and a 18-day feeding trials,the number of worm eggs output and species in each horses were calculated,and anthelmintic effects were evaluated.The results showed that we identified 9 species worm eggs,included Parascaris SP,Capillaria equouum,Cyathostomes,Triodontophorus tenuicollis,G. acgyptiacus,Strongyloides westeri,Dictyocaulus arnfieldi,Oxyuris equi and Paranoplace phala mamillana;on the 3rd day after expelling passasit, EPG was reached 13 500/g in fresh faeces;on the 6th day,the infection rate was 6.37%, the worm eggs reducing rate was 90.59%,the worm eggs flooding rate was 93.63%;On the 18th day, the infection rate was 13.33%,the worm eggs reducing rate was 71.91%,the worm eggs flooding rate was 86.67%.Therefore,there were more categories intestinal parasitic in six months old Yili horse in Xinjiang,rafoxanide had a good effect,and the effect of rafoxanide reduced after expelling passasite 18th day.

In order to understand the security of a new kind of antidiarrheal Chinese herbal medicine compound preparation for livestock,acute and sub-chronic toxicity test were conducted.Acute toxicity test used the largest drug dose method,20 Wistar rats were orally treated with the Chinese medicine compound preparation.In the sub-chronic toxicity test,80 rats were randomly divided into 4 groups with 20 rats in each group and orally given a dose of 3 000,1 500,750 and 0 mg/(kg·BW）of Chinese medicine compound preparation once a day for 30 days.The general clinical status was observed,rats weight were measured and the dose was adjusted every week during the test,after the test measured blood routine index,biochemistry index,and preceded the gross anatomy observation,weighing each major organs and calculated the viscera coefficient,and proceded main viscera histopathological observation between the high dose group and the control group.The acute toxicity results showed that every rat would be alive gavaged with the lethal dose（LD₅₀）of compound preparation larger 5 g/(kg·BW).The sub-chronic toxicity autopsy showed that except heart,lung,and testicles in individual rats appeared mild bleeding in the high dose group,the other dose group organs found no abnormal change.The haematological index showed except mononuclear cell rate（P<0.05),and hematocrit declined significantly（P<0.05）in the high dose group,all the indexes of the other groups were in the normal range,there was no significant difference from the control group.The test suggested the Chinese medicine compound preparation was no toxicity under the condition of this test according to acute toxicity classification standard of exogenous chemicals by WTO,there was no effect on the growth and development of rats in the sub-chronic toxicity test,and there was no chronic toxicity at least 1 500 mg/kg feeding conditions in short-term repeated application.

In order to understand the main bacterial pathogen species causing dairy cow mastitis in Liaoning, as well as the characteristics of drug sensitivity of the pathogenic E.coli,the milk samples from 75 dairy cows with clinical manifestations for mastitis in certain large-scale dairy farm in Liaoning were collected.The bacteria in milk were cultured and isolated with biochemical methods and in vitro drug sensitivity tests were processed with the isolated E.coli strains.The results showed that the main bacterial pathogen for dairy cow mastitis were E.coli（separation rate 58.7%),S.aureus（64.0%）and S.agalactiae（54.7%),and multiple infection including double and triple infection were identified.The drug sensitivity tests on the isolated E.coli indicated that the E.coli isolates were highly resistant to sulfonamides（resistance rate>85%）and chloramphenicol（resistance rate>30%),and they were relatively low resistant to ampicillin（9.5%),ciprofloxacin（9.5%),ceftiofur（7.1%）and ofloxacin（4.8%).The results was able to provide reliable theoretical basis for prevention and control of dairy cow mastitis in Liaoning area.