To express and predict the structural and function characteristics of the protein encoded by the truncated HSP70 gene of Theileria annulata,this study was performed on the target gene HSP70,and the gene sequence was amplified,subsequently the recombinant plasmid pMD18-T-HSP70 was constructed.Phylogenetic tree was built with the homologous HSP70 protein sequences from other HSP70 protein sequences.Bioinformatics approaches were used to analyze HSP70 protein characteristics,which included amino acids composition,basic physicochemical properties,hydrophilicity/hydrophobicity,transmembrane structure,signal peptide,possible phosphorylation sites,subcellular localization,and secondary and tertiary structures of proteins.In addition,the protein-protein interaction (PPI) network analysis of the recombinant protein HSP70 was carried out.The prokaryotic expression vector pET28a-HSP70 was constructed,and the protein expression conditions were optimized.Purification of recombinant protein was performed by His-trap affinity chromatography,and reactogenicity was tested.The results showed that the sequence of HSP70 protein of Thelieria annulata had high homology with that of Theileria parva.The molecular weight of the protein was 42 ku,the theoretical isoelectric point (pI) was 5.61,it belonged to acidic protein and hydrophilic protein,without transmembrane domain and signal peptide.Protein function prediction results showed that HSP70 contained 32 possible phosphorylation sites,the sub-cellular location analysis showed that the protein was mainly distributed in the cytoplasm.The alpha helix,beta turn,random coil and extended chain accounted for 39.18%,8.51%,30.41% and 21.91%,respectively.PPI network construction results showed that the proteins interacting with HSP70 were mainly HSP90 family members,in addition to the chaperone protein GrpE homologue,which indicated that HSP70 might form a complex with HSP90 in the cell.The prokaryotic expression vector was successfully constructed,and obtained a infusion protein with 48 ku,the optimal expression condition was at 0.6 mmol/L IPTG induced at 37 ℃ for 5 h.The results of dot blotting and Western blotting showed that the expression product could be recognized by serum of the horses naturally infected by Thelieria annulata,and the reactogenicity was ideal.The results of this experiment would provide a theoretical basis for further exploration of the functional mechanism of Thelieria annulata HSP70.

This study was aimed to clone GATA binding protein 3 (GATA3) gene in goats,construct the eukaryotic expression vector,and conduct bioinformatics analysis of GATA3 gene.The coding region(CDS) of GATA3 gene in goats was used as seed sequence (GenBank accession No.:XM_018056969.1).SnapGene 4.1.9 software was used to design primer sequence and RT-PCR method was used to amplify the complete CDS of GATA3 gene in goats.After sequencing and identification,bioinformatics analysis was conducted based on the nucleotide sequence and protein sequence,and pLVX-GATA3-IRES-ZsGreen1 lentivirus recombinant plasmid was constructed.The recombinant lentivirus plasmid pLVX-GATA3-IRES-ZsGreen1 was co-transfected with virus enveloped plasmid pCMV-VSVG and packaged plasmid pNRF into HEK-293T cells by liposome transfection method.The virus supernatant was collected after lentivirus packaging,and the ear fibroblasts in goats were successfully infected.The results of bioinformatics analysis showed that the total length of GATA3 gene CDS was 1 335 bp,444 amino acids were encoded,the molecular weight was 47.94 ku,the molecular formula was C₂₀₉₃H₃₂₃₄N₆₂₆O₆₂₅S₂₄,and the isoelectric point was 9.47,the contents of serine was the highest (13.3%),and tryptophan was the lowest (1.1%).The similarity of the nucleotide sequence of GATA3 gene in goats were 97.7%,94.2%,92.2%,92.4%,88.0% and 90.1% with Bos taurua,Sus scrofa,Equus asinus,Equus caballus,Mus musculus and Homo sapiens,respectively.The homology among different species was high and the evolution process was highly conservative.The phylogenetic tree results showed that goats were closely related to Bos taurua,Sus scrofa,Equus asinus,Equus caballus and Homo sapiens,but far related to Xenopus tropicalis and Danio rerio.Transmembrane domain and hydrophilic/hydrophobic prediction showed that GATA3 protein in goats did not exist transmembrane domain and was hydrophilic protein.The prediction of the signal peptide indicated that the protein was localized in the cytoplasm.GATA3 gene was overexpressed to produce green fluorescence signal by constructing the eukaryotic expression vector pLVX-GATA3-IRES-ZsGreen1 and transfecting with HEK-293T and ear fibroblasts in goats at the cell level.This results would provide a reference for the functional study of GATA3 gene in goats,and laid a foundation for further study on the role of GATA3 gene in lactation of goats.

The purpose of the experiment was to use bioinformatics to clone and analyze of the CDS regions of the AdipoR1 and AdipoR2 genes of the new Qinchuan beef line (hereinafter referred to as "Qinchuan beef cattle"),and to explore its expression in different tissues and muscle cells of Qinchuan beef cattle induced differentiation at different time.Taking Qinchuan beef cattle as the research object,the CDS region sequences of AdipoR1 and AdipoR2 genes were amplified by PCR,and their functional structure was predicted using bioinformatics software.At the same time,the expression level of AdipoR1 and AdipoR2 genes in 15 tissues of beef cattle and different time after induction differentiation was obtained using qRT-PCR and analyzed for differences then.The results showed that the full length of AdipoR1 gene coding sequence of Qinchuan beef cattle was 1 128 bp,encoding 375 amino acids,the protein molecular weight was 42 446.41 u,and the theoretical isoelectric point was 6.70.The secondary structure of AdipoR1 protein was mainly composed of α-helix.The prediction results of the secondary and tertiary structures were consistent.AdipoR1 protein was hydrophilic protein and had no signal peptide site.The full length of the AdipoR2 gene coding sequence was 1 161 bp,encoding 386 amino acids,the protein molecular weight was 43 707.70 u,and the theoretical isoelectric point was 6.27.The subcellular localization results showed that AdipoR1 protein was 60.9% likely to be located on the cell membrane,and AdipoR2 protein was 73.9% likely to be located on the cell membrane.The results of the amino acids phylogenetic tree of different species showed that the amino acid sequences encoded by the AdipoR1 and AdipoR2 genes of Qinchuan beef cattle were closest to Bos indicus and Bos mutus,respectively.qRT-PCR results showed that AdipoR1 and AdipoR2 genes were all expressed in 15 tissues of Qinchuan beef cattle such as heart,liver,muscle,and so on.And the expression level in muscle tissues was the highest,which was significantly higher than other tissues (P<0.01).There were also obvious differences in the timing of muscle cell differentiation.The experiment revealed the differences in the expression of AdipoR1 and AdipoR2 genes in different tissues and muscle cells of Qinchuan beef cattle at different time after induction differentiation,in order to lay a foundation for further exploration of the biological functions and regulatory mechanisms of AdipoR1 and AdipoR2 genes.

The purpose of this study was to perform cloning and bioinformatics analysis of FK506 binding protein 5(FKBP5) gene in chickens,and detect its expression of different tissues in chickens.The spleen tissue cDNA in broiler was used as a template,the complete CDS region of FKBP5 gene in broilers was amplified and cloned,the homology was compared and phylogenetic tree was constructed.The physical and chemical properties,hydrophobicity,transmembrane structure,signal peptide,secondary structure and tertiary structure of FKBP5 protein were analyzed by online software.Real-time quantitative PCR was used to detect the expression of FKBP5 gene in different tissues of valgus-varus deformity(VVD) broilers and normal broilers.The results showed that the total CDS region sequence of FKBP5 gene in chickens was 1 350 bp,which encoded 449 amino acids.The homology of amino acid sequence of FKBP5 in chickens with Coturnix japonica,Anas platyrhynchos,Gekko japonicus,Pelodiscus sinensis,Mus musculus,Homo sapiens,Sus scrofa and Danio rerio were 98.4%,96.2%,85.8%,87.4%,81.1%,85.2%,86.1% and 61.2%,respectively.The genetic relationship with Coturnix japonica and Anas platyrhynchos was the closest,followed by reptiles and mammals,and it had the farthest relationship with Danio rerio.The molecular weight of chicken FKBP5 protein was 50.43 ku,its theoretical isoelectric point was 5.94,the half-life was 30 h,the N-terminus of the peptide chain was methionine,the instability coefficient was 23.80,belonging to the stable protein.The prediction results of transmembrane region and signal peptide showed that FKBP5 protein was no transmem brane structure and signal peptide.Domain analysis showed that the protein contained two FKBP-type peptidylprolyl isomerases and three tetrapeptide repeats (TPR).FKBP5 protein structure was mainly consisted of alpha helix (46.77%),extended chain (12.47%) and random coil (40.76%),it had strong correlation with HSPA2,PPID,STIP1,HSP90AA1 and HSP90AB1.Real-time quantitative PCR results revealed that FKBP5 gene in chickes was expressed in all tissues.The expression of tissues in VVD broilers were higher than that of normal broilers,and the expression in heart,leg muscle,bursa,thymus and cartilage was significantly different (P<0.05),the expression in spleen was extremely significantly different (P<0.01).The results of this experiment provided reference for broiler bone disease and leg healthy breeding.

To comprehend the molecular evolution of fowl adenovirus serotype 4 (FAdV-4) endemic strains,based on FAdV-4 GZ-BJ and FAdV-4 GZ-QL strains isolated in the laboratory,segmented amplification was performed on the two FAdV-4 strains,the amplified product was cloned into the vector,the plasmid was extracted,PCR and double enzyme digestion were performed,and the recombinant plasmid was screened for sequencing.The sequencing results were sequentially spliced to obtain the whole genome of FAdV-4 Guizhou strain,and sequence and genetic evolution analysis.The results showed that the lengths of two FAdV-4 Guizhou strains,GZ-BJ and GZ-QL strains,were 43 352 and 43 723 bp,were obtained by PCR fragmented amplification.respectively,and the complete genes of FAdV-4 GZ-BJ strain was 371 bp shorter than the FAdV-4 GZ-QL strain,and 6 ORFs (22K,putative 9.1 ku,u-exon,ORF17,ORF28,ORF42),and the amino acid homology of them was 57.1%.The nucleotide homology of the two FAdV-4 Guizhou strains with FAdV-4 strains in different regions at home and abroad were 88.7%-100%.The FAdV-4 two Guizhou strains and domestic isolates lacked ORF19,ORF27,ORF30 that compared with the FAdV-4 classic strain ON1.Phylogenetic tree analysis showed that the two Guizhou strains FAdV-4 GZ-BJ and FAdV-4 GZ-QL still belonged to group Ⅰ species C fowl adenovirus.This research indicated that the two Guizhou strains FAdV-4 GZ-BJ strain and FAdV-4 GZ-QL strain had evolution and mutations compared with domestic and foreign FAdV-4 strains,and FAdV-4 GZ-BJ strain had changed greatly,but not yet changing its serotype and provided a basis for exploring the molecular mechanism of FAdV-4 virus pathogenic mechanism.

The aim of this study was to analyze the effect and mechanism of miR-199a-5p on lipid production of porcine intramuscular adipocytes.The longissimus dorsi muscle and subcutaneous fat tissue of Huainan pigs were obtained at different fattening periods (before,middle and after fattening periods),and the expressional trend of miR-199a-5p was detected by Real-time quantitative PCR.mimics of miR-199a-5p was synthesized and transfected in porcine intramuscular adipocytes,the lipid deposition was detected by Oil Red O staining after differentiation.The relationship between the previously detected meat quality-related differently expressed mRNAs,lncRNAs,circRNAs and miR-199a-5p was analyzed by miRanda software,and the function of miR-199a-5p target genes (target mRNAs,co-expressed mRNAs of lncRNAs,parent genes of circRNAs) was analyzed by GO and KEGG enrichment.The results indicated that miR-199a-5p was continuously upregulated in longissimus dorsi muscle during fattening period,but it was firstly upregulated and then downregulated in subcutaneous adipose tissue.Overexpression of miR-199a-5p inhibited adipogenesis in intramuscular adipocytes.There were 9 mRNAs,5 lncRNAs,and 1 258 circRNAs owned miR-199a-5p binding sites,KEGG analysis showed that these genes were mainly enriched in sugar,lipid and protein metabolism,and GO enrichment mainly involved plasma membrane region and acting binding.The results indicated that miR-199a-5p might participate in muscle development,adipogenesis and lipid metabolism by interaction with lncRNAs and circRNAs,and it could be used as a candidate miRNA for meat quality trait.

To study the regulation role of estradiol (E₂) on long non-coding RNA (lncRNA) expression in thymic epithelial cells (TEC).The cell phenotypic changes were observed after the 50 nmol/L E₂ was added in cultured mouse epithelial cell line 1 (MTEC1),while the cell viability was also measured with CCK-8 kit.Furthermore,total RNA was extracted and the expression regulatory role of E₂ on lncRNA-2410006H16Rik was verified by Real-time quantitative PCR.Moreover,the target gene was amplified by RT-PCR to construct a recombinant vector of pEGFP-N1-lncRNA-2410006H16Rik.The results showed the proliferation of MTEC1 cells was significantly inhibited by 50 nmol/L E₂,and the D_{450 nm} value was also extremely significantly reduced as compared with control group detected by CCK-8 (P<0.01),indicating that the cell viability was extremely significantly decreased by E₂.Real-time quantitative PCR results indicated that the expression of lncRNA-2410006H16Rik in MTEC1 cells was extremely significantly up-regulated by 50 nmol/L E₂ (P<0.01),and about two fold-changes higher than control group cells,which was consistent with the high-throughput sequencing results.RT-PCR,double enzyme digestion and sequencing results showed that the pEGFP-N1-lncRNA-2410006H16Rik expression vector was successfully constructed.All the above results indicated that the expression of lncRNA-2410006H16Rik in TECs was closely related to the effect of E₂,which laid a foundation for further verification of the regulatory function of lncRNA-2410006H16Rik at the cellular level.

The purpose of this experiment was to clone the Siberian tiger γ-interferon (IFN-γ) gene,studied its molecular characteristics and predicted the biological function of the protein,making preliminary preparations for the subsequent study of the antiviral activity of interferon.The Siberian tiger IFN-γ gene was amplified from ConA-induced Siberian tiger blood lymphocytes by RT-PCR and sequenced to obtain the sequence.Bioinformatics was used for sequence analysis.The results showed that IFN-γ gene coding region consisted of 504 nucleotides,encoding a total of 167 amino acids.The relative molecular weight of the Siberian tiger IFN-γ amino acid was 19.59 ku,the isoelectric point was 9.03.The encoded protein was a basic hydrophilic protein,of which the first 23 amino acids might be signal peptides.The conserved domain of the IFN-γ encoded protein was the IFN-γ superfamily,and there was a transmembrane structure,in which amino acids 1－6 were intracellular regions and amino acids 7－28 were transmembrane regions,amino acids 29－167 were extracellular regions.The secondary structure of IFN-γ encoded protein was mainly α-helix (58.08%) and random coil (33.53%).There were 5 potential B cell epitopes and 3 potential N-glycosylation sites.Sequence analysis showed that the Siberian tiger IFN-γ had 80.4%－99.8% nucleotide similarity to the Siberian tiger,African lion,leopard,puma,cheetah,domestic cat,Canadian lynx and wild boar published on GenBank,and the amino acid similarity was 70.5%－100%,nucleotide genetic evolution analysis showed that Siberian tiger IFN-γ had the closest relationship with African lions and leopards,followed by puma,cheetah,domestic cat and lynx,and wild boars were farthest.By synthesizing the modified Siberian tiger IFN-γ gene,a recombinant plasmid pPIC9K-IFN-γ capable of secreting and expressing IFN-γ protein was constructed,which was introduced into a highly efficient expression system-Pichia pastoris to induce expression,and analyzed by SDS-PAGE,the molecular weight of the expressed protein was about 17.8 ku,which was consistent with the expected size,indicating that the Siberian tiger IFN-γ was successfully expressed.

The aim of this study was to find out the replication of Pseudorabies virus (PRV) in cell lines with NF-κB family the p65 gene knocked out.The 3D4/21 cell lines with p65 gene stable knocked out were constructed by site-specific gene modification techniques CRISPR/Cas9.The recombinant plasmid p65-sgRNA was constructed and transfected into HEK293T/17 cells.Lentivirus was collected and infect 3D4/21 cells.The polyclonal cell lines was gained by puromycin screening.The knockout efficiency was detected by T7 nuclease digestion.The stable cell line of 3D4/21-p65^-/- was obtained by finite dilution method.The effect of p65 gene knockout on cell proliferation in 3D4/21 cell lines was detected by CCK-8 kit.The difference of virus proliferation in 3D4/21 and 3D4/21-p65^-/- was detected by flow cytometry.The mRNA expression level of PRV gB,TK,IL-1β and IL-6 gene were detected by Real-time quantitative PCR after infecting PRV-QXX in 3D4/21 and 3D4/21-p65^-/- cells.The protein expression of PRV gB and gE were detected by Western blotting after infecting PRV-QXX in 3D4/21 and 3D4/21-p65^-/- cells.The titration of the progeny PRV-QXX produced by infecting PRV-QXX in 3D4/21 and 3D4/21-p65^-/- cells was measured by traditional plaque formation assay and developed median tissue culture infective dose(TCID₅₀)methods.The results showed that the gene editing efficiency of sgRNA2 and sgRNA3 was higher.The stable expression cell lines with p65 gene knockout were obtained by cloning and culturing.CCK-8 kit test was used to detect cell viability,result showed that p65 gene knockout had no effect on it.The result of flow cytometry detection at the same time point showed that the proliferation of PRV-GFP in 3D4/21-p65^-/- cells was significantly higher than that in control cells.The result of Real-time quantitative PCR was exhibit that in 3D4/21 cells,p65 gene knockout promoted the mRNA expression of PRV gB and TK gene,but IL-1β and IL-6 genes were inhibited.Western blotting was used to demonstrate that p65 gene knockout promoted the expression of PRV gB and gE protein in 3D4/21 cells.The virus titer result gene showed that the replication of PRV-QXX was significantly higher than that of control cells in 3D4/21-p65^-/- at the same time.The above results suggested that p65 gene knockout promoted PRV replication in 3D4/21 cells.

In order to reveal the composition of fishmeal and ensure the quality of fishmeal,high-throughput sequencing technology was used to sequence 5 imported fishmeal samples from different countries,and the composition of fishmeal derived from fish was analyzed based on COI gene and its abundance.After DNA was extracted from fishmeal samples,fish COI gene amplification primers were used for PCR amplification to obtain fish mixed COI gene sequence products.The amplified products were sent to the sequencing company for high-throughput sequencing.The COI gene sequencing data were compared with the fish related databases,and the species-derived components of fishmeal were analyzed through the comparison results.The results showed that 5 fishmeal samples were successfully obtained,and the main component of white fishmeal was cod.The composition of red fishmeal was diverse,including herring,swordfish and so on.The data were statistically analyzed by Python,and the COI gene abundance map of fish after PCR amplification was drawn to further intuitively indicate the composition of raw materials in fishmeal samples.The results showed that high-throughput sequencing technology could be used in the quality evaluation of fishmeal,which was helpful to quickly understand the composition of fishmeal and guide its quality and use.At the same time,it was suggested that this technology could be further applied to the monitoring of fishmeal adulteration and pathogenic microorganisms,which was of great significance to ensure the quality of fishmeal products and maintain the normal market order.

The purpose of this experiment was to study the effect of modifying transport vehicles on alleviating donkey transport stress.28 healthy Dezhou male donkeys were selected and randomly divided into 2 groups,which were transported from Beijing Miyun county to Liaocheng Donge Black Donkey Research Institute.The distance was about 500 km and the transportation lasted 18 h.9.6 m single-deck trucks were used.The modified transport vehicle which had the top floor and bilateral tarpaulins,straw curtain at the bottom and partition in the middle was used in experimental group(T).The untreated vehicle was used in the control group (C).The blood biochemical and hormone indexes were measured,the change of body weight and the number of donkeys infected were counted.The results showed that:Compared with before transportation,the serum indexes of cortisol (COR),albumin (ALB) were significantly higher on the day of landing (P<0.05),total protein (TP),aspartate aminotransferase (AST),heat shock protein-90 (HSP90) and creatine kinase (CK) indexes were extremely significantly increased (P<0.01),calcium (Ca) concentration was significantly decreased (P<0.05) in contron group.Compared with ordinary vehicle group,the indicators such as TP,AST and CK range of variation of the modified vehicle group were lower.The blood glucose (GLU) were increased after landing in both groups compared with before transportation and with no significant difference (P<0.05).Compared with control group,the average body weight change after transportation of the donkeys in modified vehicle group was lightly.On the 10th day after landing,the average weight of the donkeys in modified vehicles was close to that before transportation,while the average weight of the donkeys in control group was basically restored as before transportation on the 20th day after landing.It means that the use of the modified vehicles could significantly promote weight recovery.Statistics of the donkey's morbidity and mortality of the two groups indicated that the incidence of donkeys was 21.4% in control group and 7.1% in modified vehicles.No donkey died in both two groups.The results showed that the transformation of transport vehicle affected the stress of donkeys,which was of great significance to the protection of their health,and could provide reference for increasing animal welfare and reducing economic losses against tranportation stress.

The goal of this study was to elucidate the impact of low environment temperature on the performance of Dorper sheep and Suffolk sheep,and to further reveal the internal mechanism,provide for hybridization using data reference in Inner Mongolia.Twenty 12-month-old healthy Dorper sheep,Suffolk sheep and Mongolian sheep with similar body weight (half male and half female) were selected,and the latter was used as control.The physiological and serum biochemical indicators,and immune performance were measured and compared.The results showed that the body temperature,respiratory rate and heart rate in a whole day were not significantly different between Dorper and Suffolk sheep (P>0.05),but their heart rates were lower than the Mongolian sheep (P<0.05).The body temperature of Dorper sheep was significantly lower than Mongolian sheep in the morning (P<0.05),and the respiratory rate was significantly higher than Mongolian sheep all day (P<0.05).Compared with Mongolian sheep,the activities of serum alanine aminotransferase (ALT),superoxide dismutase (SOD) and the levels of triiodothyronine (T₃) and thyroxine (T₄) in Dorper sheep and Suffolk sheep had no significant difference (P>0.05),while aspartate aminotransferase (AST) and alkaline phosphatase (AKP) activities were significantly higher (P<0.05).The activity of lactate dehydrogenase (LDH) in Suffolk sheep was significantly higher than Duper sheep and Mongolian sheep (P<0.05).The levels of interleukin 2 (IL-2),interleukin 4 (IL-4),immunoglobulin A (IgA),immunoglobulin M (IgM) and immunoglobulin G (IgG)in Dorper sheep and Suffolk sheep serum were not significantly different compared with Mongolian sheep (P>0.05),but the concentration of serum IL-2 in Dorper sheep was significantly higher than Suffolk sheep (P<0.05).In conclusion,under the low environment temperature in Inner Mongoliar,the heart rate of both Dorper sheep and Suffolk sheep throughout the day were significantly lower than Mongolian sheep,and the activities of ALT and AKP in serum were significantly higher than Mongolian sheep.The serum LDH content of Dorper sheep was significantly lower than Suffolk sheep,and the content of IL-2 was significantly higher than Suffolk sheep.Except for AST,the other serum biochemical indicators in experimental sheep were within the normal range.It showed that in the low temperature environment of Inner Mongolia,Dorper sheep and Suffolk sheep could maintain normal physiological conditions and were better adapted to the environmental climate in Inner Mongolia.

Breast cancer is a common disease in dogs,cats and other companion animals and humans.As a malignant tumor and the main cause of death of humans and animals,the disease burden of breast cancer is still gradually increasing,and the prevention and treatment situation of breast cancer is becoming more and more serious.Epithelial-mesenchymal transition (EMT) plays an important role in biological process in the development of breast cancer,which can also promote the invasion,metastasis and drug resistance of malignant tumors.Therefore,it is getting more and more attention in cancer research,and targeting EMT is an important research direction and hotspot in the treatment of breast cancer.In this paper,the changes of cell morphology and function and markers,the classification of EMT and the relationship between EMT and breast cancer were discussed respectively.The EMT-related TGF-β/Smad,NF-κB and Wnt signaling pathways were analyzed in detail.And then advances in the treatment of breast cancer,including the development of inhibitors of TGF-β/Smad pathway,the prospect of treatment with related drugs,genes and cytokines,and the experimental results of NF-κB pathway and Wnt pathway inhibitors,were discussed in detail.Finally,the development and trend of the treatment of breast cancer were prospected.Deeply understanding the biological process of breast cancer EMT regulated by signaling pathway,making clear its occurrence development mechanism,finding key targets and developing targeted drugs,will bring the dawn for the precise treatment of breast cancer.

This experiment was conducted to determine isoleucine (Ile) requirement of Yellow-feathered broiler chickens at different feeding stages.2 400,2 160 and 1 920 Yellow-feathered broiler chickens of 0,22 and 43 days age were selected,according to gender and body weight,they were randomly divided into eight dietary treatments with six replicates (half male and half female),there were 50,45 and 40 chickens at 0-21,22-42 and 43-63 days of age,the levels of Ile in basal diet were 0.55%,0.50% and 0.45%,respectively.Ile was added gradiently in the basal diet in each treatment group,and the inter group interval level was 0.05%,the nutritional levels of all treatments were the same except for Ile.The experimental chickens were raised on the ground,and they were free to eat granular materials and drink freely.The results showed that the level of Ile and gender had significant effects on the growth performance of the three stages,and their interaction had significant effects on growth performance during the three stage (P<0.05).The average daily gain (ADG) and feed/gain (F/G) showed significant or extremely significant quadratic response with the increase of dietary Ile level (P<0.05;P<0.01).The prediction results of the two-slope broken-line model showed that,at 0-21 days of age,the optimal dietary Ile level that maximized ADG were 0.77%,0.72% and 0.73%,that optimized F/G were 0.75%,0.70% and 0.73% for male,female and mix chicks,respectively.At 22-42 days of age,the optimal dietary Ile level that maximized ADG were 0.68%,0.64% and 0.66%,that optimized F/G were 0.66%,0.62% and 0.64% for male,female and mix chicks,respectively.At 43-63 days of age,the optimal dietary Ile level that maximized ADG were 0.64%,0.59% and 0.61%,that optimized F/G were 0.61%,0.57% and 0.59% for male,female and mix chicks,respectively.In conclusion,the Ile requirement decreased with the increase of age,its requirement of male was significantly higher than that of female,and the nutrient requirement of Ile evaluated by F/G was lower than the recommended value based on ADG.

In order to evaluate amino acids (AA) requirement of Wanxi White goose from 9 to 12 weeks of age,eighty-four Wanxi White geese at 9 and sixteen adult Wanxi White male geese were used in a comparative slaughter trail and a nitrogen balance trail,respectively,based on the principle of factorial method.Carcass and feather from geese at 9 weeks of age and 12 weeks of age and excretion from adult male geese were collected for AA analysis.In addition,excretion from adult geese was used for amino acid and creatinine analysis.The results were as follows:There were no significant differences in the AA composition of carcass and feathers between geese at 9 weeks of age and 12 weeks of age (P>0.05).The requirements (the number in brackets was AA pattern) of lysine,methionine,cysteine,threonine,histidine,valine,leucine,isoleucine,arginine,phenylalanine,serine,and proline for growth in geese from 9 to 12 weeks of age were 1 803.16 (100),464.85 (26),775.56 (69),1 032.40 (57),482.37 (27),1 200.68 (67),1 862.72 (103),1 043.63 (58),1 203.58 (67),660.97 (37),1 322.75 (73),and 824.05 (46) mg/d,respectively,and those for maintenance were 172.29 (100),161.14 (94),633.05 (367),398.08 (231),70.52 (41),548.26 (318),626.06 (363),362.46 (210),506.62 (294),299.31 (174),882.53 (477),and 693.37 (402) mg/d,respectively.Dietary levels of lysine,methionine,cysteine,threonine,histidine,valine,leucine,isoleucine,arginine,phenylalanine serine,and proline in Wanxi White geese from 9 to 12 weeks of age were 0.77%,0.24%,0.55%,0.56%,0.21%,0.68%,0.97%,0.55%,0.66%,0.37%,0.83%,and 0.59%,respectively.

This study aimed to evaluate the variation in rumen fermentation and microbiota of pre- and post- early weaning lambs so as to provide theoretical basis for understanding the rumen development and early breeding of lambs.Sixteen male lambs with similar birth weight (BW,3.81 kg±0.55 kg) were selected.Lambs were fed breast milk from 1 to 7 days old.The lambs were separated from their dams at 8 days old and began to be fed milk replacer (MR) (at 2% of BW measured on day 8,three equal amounts) and starter feeds (ad libitum).MR feeding was stopped at 35 days of age.Six lambs were slaughtered pre-weaning (21 days old) and post-weaning (42 days old) respectively and rumen contents were collected to determine rumen fermentation,enzyme activity and microbiota.The results showed that the total volatile fatty acids,acetic acid,propionic acid,butyric acid,activities of cellulase and α-amylase for post-weaning lambs were higher than those for pre-weaning lambs (P<0.01).The diversities and abundances of rumen flora for post-weaning lambs were lower than those for pre-weaning lambs (P<0.05).The dominant phylum for pre- and post-weaning lambs were Bacteroidetes and Firmicutes,in which Bacteroidetes was the first phylum,accounting for 61.96% and 65.36% in pre- and post-weaning periods respectively.And Firmicutes was the second phylum,accounting for 32.08% and 24.03% in pre- and post-weaning periods respectively.The total of the two phylum accounted for 94.04% and 89.39% for pre-weaning and post-weaning lambs.The dominant genus for pre- and post-weaning lambs were unidentified_Prevotellaceae,accounting for 21.85% and 38.49% respectively.There was no significant change in rumen microbial function for pre- and post-weaning lambs,which all mainly focused on replication and repair,carbohydrate metabolism and translation.These results indicated that the rumen fermentation and enzyme activity were increased for post-weaning lambs,whereas the diversity and abundances of flora were decreased,the dominant flora and the function were similar between pre- and post-weaning lambs.

This experiment was conducted to investigate the effects of metabolizable lipogenic substances (MLS) on the growth performance,carcass quality and meat quality of growing pigs.Eighteen healthy Duroc×Landrace×Yorkshire hybrid pigs with similar body size and body weight range of 28.15 kg±4.51 kg were randomly divided into three groups.The diets with the same nutritional level but different MLS levels were fed for 28 days.MLS levels were 70.51 g/d (group Ⅰ),68.09 g/d (group Ⅱ) and 40.58 g/d (group Ⅲ).The growth performance,carcass quality and meat quality of the growing pigs were measured.The results showed that the effects of different MLS levels on average daily feed intake,average daily gain and feed and gain ratio of pigs were not significant (P>0.05).Among the three groups,feed and gain ratio of group Ⅱ was the lowest.There was no significant difference in carcass indexes such as slaughter rate,backfat thickness and eye muscle area among the three groups (P>0.05).The cooked meat rate of group Ⅲ was significantly lower than groups Ⅰ and Ⅱ (P<0.05),while other meat quality indexes such as brightness value,marble score and dripping loss had no significant difference (P>0.05).There was little difference in pH between 45 minutes and 24 hours after slaughter between groups (P>0.05).In conclusion,under the conditions of this experiment,most indexes of growth performance,carcass quality and meat quality of growing pigs were not significantly different among those of three metabolizable lipogenic substances level diets,but they were of guiding significance for optimizing the formula and future production.

The experiment was conducted to study the effects of adding different levels of Eucommia ulmoides leaves (EUL) powder on egg quality,serum biochemical indexes,slaughter performance and meat quality of Green-shell layers.80 healthy Green-shell layers with the similar body weight,egg-production rates and good healthy conditions were randomly divided into 4 groups with each group 20 replicates and each replicate had 1 chicken.Each group was fed corn-soybean meal diet supplemented with 0% (CTL group),2% (EUL1 group),4% (EUL2),6% (EUL3) Eucommia ulmoides leaves meal.The trial period lasted for 5 d,the formal experiment period lasted for 70 d.The egg quality,serum biochemical indexes,slaughter performance and meat quality of each group of chickens were measured after the feeding test.The results showed as follows:Adding Eucommia ulmoides leaves powder into diets could significantly decreased the triglyceride (EUL1,EUL2,EUL3),glucose (EUL2,EUL3) and urea (EUL2,EUL3) in serum (P<0.01).There was no significant difference in egg quality and slaughter performance (P>0.05).Liver weight (EUL1,EUL2) and heart weight (EUL1,EUL2,EUL3) were significantly reduced (P<0.01).Thigh muscle protein (EUL1) and pectoral muscle fat (EUL2) content were significantly increased (P<0.05).The pH_{24 h} of the leg muscles (EUL2) was significantly increased (P<0.05).The shear force of the leg muscle (EUL1) was significantly reduced (P<0.01).In conclusion,Eucommia ulmoides leaves powder could significantly reduce the levels of triglyceride,glucose and urea in serum.There was no significantly difference in egg quality,but the muscle quality of Green-shell layers were improved,pectoral fat leg muscle protein content and leg muscle tenderness were increased,liver weight and heart weight were significantly reduced.In this experiment,the suitable adding level of Eucommia ulmoides leaves powder in layer feed was 4%.

The aim of this experiment was to study the effect of inosine monophosphate acid (IMP) on egg performance,egg quality and hatching performance in breeding chickens.864 healthy AA breeds at 20 weeks old with the same genetic background and similar body weight,and with an egg laying rate of 5% were choosed and randomly divided into 2 groups (control and test groups,fed with basic diet and basic diet＋0.5% IMP,respectively.) with 6 repetitions and 72 replicates each.The experiment lasted 30 days.The average egg weight,egg laying rate and qualified egg rate of all kinds of eggs were measured during the test period,and some eggs were randomly selected for egg quality determination in each group.On the 17th day of entering the hatchery,the egg fertilization rate was counted by egg candling,and the hatching rate,brooding rate and healthy brooding rate were calculated on the 21st day.The results showed that:Compared with the control group,there was a tendency to promote average egg weight,laying rate and qualified egg rate in the test group,but the difference was not significant (P>0.05).The egg shape index of the egg quality in the test group was significantly increased (P<0.05),and the eggshell strength,egg shell thickness and egg yolk color of the egg quality were significantly decreased (P<0.05).The fertilization rate,hatching rate of fertilized eggs and hatching rate of hatching eggs in the experimental group were significantly increased (P<0.05).The healthy brooding rate and the weight of the chicks were increased,but the difference was not significant (P>0.05).In conclusion,0.5% exogenous IMP could promote egg laying performance and egg hatching performance of breeding chickens.

Copper plays a very important role in ruminants,which is an essential trace element for maintaining normal physiological function,biochemical metabolism and growth of ruminants,many enzymes that depend on copper play an important role in ruminants,due to copper deficiency in the herbage of Qinghai-Tibet plateau in cold season,the grazing livestock tended to suffer from copper deficiency,which affects the growth and the improvement of production performance of grazing livestock,for this purpose,a certain amount of copper is needed for grazing livestock,but copper has a low absorption rate in ruminants and affected by many factors,these factors should be researched to improve the absorption efficiency,and lower pollution to the environment.Copper promotes rumen microbial growth,improve the efficiency of rumen fermentation and nutrient digestibility by increasing gas production and energy utilization efficiency,it can also improve the immune function and antioxidant performance of ruminants,eventually copper can improve ruminant production performance,different ruminants or the same ruminants at different physiological stages have different copper requirements,copper should be added according to the nutrient requirements and combined with the actual production.This paper reviewed copper absorption and its affect factors,and the effects of copper on rumen fermentation,nutrient digestibility,immune function,antioxidant performance,production performance of ruminants,in order to provide reference for application of copper in ruminants.

The purpose of this study was to explore the possible association of the intestinal microbiome with early-stage weight in Duroc pigs.80 days old weaned piglets with Lighter body weight (LBW,n=9) and heavier body weight (HBW,n=9) were selected from 91 individuals based on their body weights ranked.Fecal samples from piglets in the early grower phase were performed for microbial diversity,composition and predicted functionality by sequencing the V3-V4 region of 16S rRNA gene.The results showed that there was no significant differences in microbial Alpha diversity between HBW and LBW groups(P>0.05).The two dominant phyla detected in both groups were Firmicutes (65.62% and 66.57% in HBW and LBW groups) and Bacteroidetes (30.87% and 29.80% in HBW and LBW groups).Four dominant genera were Prevotella (23.82% and 20.84% in HBW and LBW groups),Lactobacillus (9.11% and 16.55% in HBW and LBW groups),norank_f_Ruminococcaceae (10.32% and 9.98% in HBW and LBW groups),and norank_f_Lachnospiraceae (6.33% and 4.53% in HBW and LBW groups).PCoA analysis showed that there were small differences in gut microbes among Duroc piglets,whereas,some weight-associated microbial compositional differences were revealed between HBW and LBW groups.At the genus level,the abundance of several genera,such as g_Kitasatospora,g_norank_o_Tremblayales,g_norank_f_Erysipelotrichaceae,g_unclassified_f_[Paraprevotellaceae] and g_Coprobacillus,were significantly higher in HBW group than in LBW group (P<0.05).The abundance of Corynebacterium in LBW group was significantly higher than that in HBW group (P<0.05).Moreover,the KEGG pathways analysis showed that the sesquiterpenoid biosynthesis,insulin signaling pathway,ascorbate and aldarate metabolism,and biosynthesis of ansamycins were significantly enriched in HBW group (P<0.05),however,the secretion system pathway was significantly enriched in LBW group (P<0.05).These results might provide insights into host-microbe interactions occurring in the early stage piglets and would be useful in contributing to healthy pig breeding.

The objective of this study was to explore general characteristics of individual culling and analysis the influencing factors of longevity in Holstein cows in Ningxia.Descriptive statistics for productive life,herd life and lactation number in 15 523 Holstein cows were obtained,and culling characteristics under different culling parities,culling seasons and culling lactation stages were analyzed.Secondly,phenotypic correlations among productive life,herd life and lactation number were calculated,and a fixed model was adopted to assess the impacts of herd-birth year,birth season,herd size,culling reason and first calving age on longevity of Holstein cows.The results showed that the average utilized parity was about 2.36,the average productive life of Holstein cows was 736.59 d in Ningxia.There was the highest culling risk in the first month after birth of Holstein cows.With the increase of lactation stage,culling risk of cows gradually decreased within each lactation.With the increase of the culling parity,the culling of dairy cows was more concentrated in the early stage of postpartum lactation.There were high phenotypic correlations (>0.9) among productive life,herd life and lactation.Herd birth year,birth season,herd scale,culling reason and the first calving age had significant effects on longevity (P<0.05).This study initially revealed culling characteristics of Holstein cows and influencing factors of longevity in Ningxia,which laid the foundation for the selection of longevity traits in Holstein population in Ningxia.

In order to explore the mechanism of myostatin (MSTN) on the growth and development of bovine skeletal muscle,this study aimed at the expression of decorin (DCN) with large fold difference obtained by quantitative proteomic and phosphoproteomic screening of muscle tissues of MSTN^+/- Mongolian cattle and wild Mongolian calf gluteal muscle,and isolated and cultured bovine skeletal muscle satellite cells in the pre-laboratory.The model of induced myogenic differentiation in vitro was established as the object.Through screening the interference effects of three DCN siRNAs designed and synthesized,the most significant interference effect of si-DCN-2 (si-DCN) was transfected into bovine skeletal muscle satellite cells.Real-time fluorescence quantitative PCR and Western blotting were used to detect the changes in the expression of the proliferation markers Pax7 and MyoD in proliferative phase (GM) bovine skeletal muscle satellite cells and the effects of interfering DCN on cell proliferation was detected using EdU staining.In vitro myogenic differentiation of bovine skeletal muscle satellite cells transfected with DCN siRNA was performed.Myotube formation on the third day of differentiation (DM3) of bovine skeletal muscle satellite cells was observed by microscopy,and Real-time fluorescence quantitative PCR and Western blotting were used to detecte the expression changes of the differentiation markers MyoG and MyHC at the mRNA level and protein level,and myotube MyHC at DM3 stage was stained by immunofluorescence to study the effect of interfering with DCN on cell differentiation.The results showed that after interfering with DCN expression,the mRNA and protein levels of Pax7 and MyoD in bovine skeletal muscle satellite cells in the proliferative phase were significantly or extremely significantly upregulated (P<0.05;P<0.01),and the rate of EdU-positive cells was significantly increased (P<0.05),indicating that interfering with DCN expression significantly promoted the proliferation of bovine skeletal muscle satellite cells.After interfering with the expression of DCN,the diameter of myotubes induced by bovine skeletal muscle satellite cells on the third day of differentiation showed an increasing trend.The expression of MyoG,a marker of myogenic differentiation,was extremely significantly and significantly higher than that of the control group at the mRNA and protein levels respectively (P<0.01;P<0.05).MyHC was significantly lower at the mRNA level (P<0.05),but significantly higher at the protein level (P<0.01).Immunofluorescence results showed that the myotube fusion index was significantly higher after downregulation of DCN than that of the control group (P<0.05),indicating that interfering with DCN expression can promote the myogenic differentiation process of bovine skeletal muscle satellite cells.The results of this study showed that interfering with DCN could significantly promote the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells.The results laid a foundation for further research on the regulatory mechanism of MSTN on myogenic differentiation of bovine skeletal muscle satellite cells.

With the development of molecular genetics,a large number of quantitative trait sites and candidate genes that affect residual feed intake (RFI) have been identified.Mitogen-activated protein kinase 5 (MAP3K5),also known as apoptosis signal-regulating kinase 1 (ASK1),is one of the MAPK super family genes.Currently,there are extracellula signal-regulated protein kinase (ERK),c-Jun N-terminal kinase (JNK) and p38 mitogen-active protein kinase (p38-MAPK) the three members of the MAPK family were cloned and identified in mammalian cells,and their main biological mechanism was to mediate three MAPKs signaling pathways,thus affecting the growth,body size and milk production traits of livestock.In previous study of the residual feed intake in cattle,we screened out the MAP3K5 gene related to cattle RFI.Although the function of the MAP3K5 gene in cattle was not clear currently.Based on this,this article reviewed the structure and biological function of the gene,summarized the function and role of the gene in the main livestock and poultry feed intake variation and human obesity phenotype,and analyzed the MAP3K5 gene from the perspective of genetics.Possible mechanisms in the regulation of RFI phenotype in livestock and poultry.By reviewing the research progress of MAP3K5 gene in the regulation of livestock and poultry RFI phenotype,it was hoped to provide ideas for further research on the molecular mechanism of MAP3K5 gene in the regulation of livestock and poultry feed intake traits.For other possible effects of MAP3K5 gene on livestock and poultry phenotypic factors (such as intestinal flora) need to be further explored.

The aim of this study was to detect the transcriptional differences of 5-hydroxytryptamine N-acetyltransferase (AANAT) gene in ovine ovarian tissue during the anestrus and breeding season (follicular stage and luteal stage) and to analyze whether the transcriptional differences were caused by changes in the degree of DNA methylation modification.In the study,non-pregnant Tan sheep whose natural environmental conditions and feeding management were consistent and the weight difference was within 0.5 kg were selected.Their ovarian tissues during the anestrus,follicular and luteal stages (3 ewes in each stage) were collected.SYBR dye method was used to carry out Quantitative real-time PCR to detect the transcription level of AANAT gene in the ovarian tissue of tan sheep at different reproductive stages.Subsequently,MethPrimer 2.0 online software was used to predict the CpG island in promoter region and the first exon region of AANAT gene,and the bisulfite sequencing (BSP) method was used to detect the methylation degree of the promoter region and the first exon region of the AANAT gene for the anestrus and follicle stage samples with transcriptional differences.The results showed that the transcriptional level of AANAT gene in the ovarian tissue of Tan sheep during anestrus was significantly lower than that in the follicular period in the breeding season (P<0.05),and there was no significant difference of transcriptional level between anestrus and luteal period (P>0.05).There was a CpG island with a length of 173 bp in the AANAT gene promoter region and a CG island with a length of 118 bp in the first exon region in the Tan sheep ovarian tissue.However there was no significant difference on the methylation degree of the single CpG site in promoter region and the first exon of the AANAT gene in Tan sheep ovary tissue,suggesting that the expression difference of the AANAT gene was regulated by other factors other than methylation modification.The results of this study might provide references for further study on the function of AANAT gene in seasonal estrus and follicular maturation.

In the process of eukaryotic genome transcription,in addition to some protein coding RNAs,there are some different types of non-coding RNA (ncRNA) also participate in the regulation of complex life processes.In recent years,the emergence of whole transcriptome sequencing technology and the rapid discovery of bioinformatics have greatly promoted the research of ncRNA,revealing the important role of ncRNA in various aspects of the biological field.Including the treatment of tumor and other non tumor diseases in human,the development of embryo,muscle growth,fat deposition,immune response and regulation of skin hair follicle in animals have made great progress.The author mainly introduced the whole genome sequencing technology,the classification of ncRNA,the main mechanism of action and the application of ncRNA in livestock and poultry,it provided a theoretical basis for exploring the regulatory mechanism of ncRNAs in animals.

Hair follicles are the basis for the growth of hair fibers,and determine the properties of wool fibers.Keratin is one of the important components of hair follicles.In recent years,some progresses have been made in hair follicle growth and development and the mining of keratin genes.The article reviewed the discovery of keratin genes,which were already equivalent to human keratin genes,but there were differences in number and classification.This paper introduced the morphological structure of hair follicles,briefly described the gene expression and localization of hair follicles keratin gene and the keratin influencing the character of hair fibers.The important effect of proteomics technology in hair follicle on the growth and development of hair follicle and fiber character was analyzed,in order to provide a new research idea for the growth and development of sheep hair follicles and the molecular breeding of wool traits.

In order to explore the role of the type Ⅳ secretion system in the process of Brucella infection,in-depth understanding of the potential of the type Ⅳ secretion system of Brucella in vaccine development,in this study,the A19 vaccine strain of Brucella abortus was used as the research object,and the A19 VirB promoter deletion strain was used to infect mouse dendritic cells (DCs),evaluate the effect of type Ⅳ secretion system on the adhesion,invasion and intracellular survival of Brucella by colony count (CFU).At the same time,RNA and total protein were extracted from the infected cells,and the transcription and expression of the autophagy gene Beclin-1 were detected by Real-time PCR and Western blotting respectively.The cell supernatant after infection was collected,and ELISA was used to detect the secretion levels of inflammatory factors IL-6 and IL-10.The adhesion and invasion results showed that the adhesion and invasion levels of Brucella VirB promoter deletion strain and parent strain A19 were not significantly different (P>0.05).The intracellular survival experiment found that the intracellular survival ability of the Brucella VirB promoter deletion strain was significantly lower than that of the parent strain A19 at 4 h after infection (P<0.05),and it was extremely significantly lower at 0,24 and 48 h after infection (P<0.01).The results of Real-time PCR and Western blotting showed that the level of Beclin-1 production was extremely significantly higher than that of the parent strain A19 (P<0.01).ELISA results showed that at 8 and 12 h after infection,the level of IL-6 stimulated by VirB promoter deletion strain was significantly higher than that of parental strain A19 (P<0.05).However,at 8 and 12 h after infection,the level of IL-10 stimulated by VirB promoter deletion strain was significantly lower than that of parental strain A19 (P<0.05),and was extremely significantly lower than that of parental strain A19 at 24 h after infection (P<0.01).This study preliminarily explored the biological role of Brucella type Ⅳ secretion system in the process of infection of DCs,and laid a theoretical foundation for the follow-up Brucella vaccine transformation research.

The purpose of this study was to obtain the HNex protein of Bovine parainfluenza virus (BPIV3) and its polyclonal antibody.The extracted BPIV3 cytotoxic RNA was used as the template,and the gene fragment containing HN was amplified by RT-PCR,and then using this as the template,the HN protein outer region (HNex gene) was amplified and the nucleotide sequence was determined.The HNex gene was inserted into the cloning vector pEASY-Blunt Simple,and then double-digested into the pET-30a(+) expression vector to construct a recombinant prokaryotic expression vector pET-BPIV3-HNex and transformed into competent Rosetta^TM(DE3) pLysS.After IPTG induction,the expression products were identified by SDS-PAGE and Western blotting.HNex protein purified by affinity chromatography was used as immunogen to prepare rabbit anti-BPIV3-HNex polyclonal antibody.Sequencing results showed that this study successfully cloned the BPIV3 HNex gene.The prokaryotic expressed protein results showed that a specific band appeared at the 53 ku position,and it could specifically react with murine anti-His.The ELISA titer of BPIV3-HNex antibody in the serum of immunized rabbits was 1:819 200.The Western blotting results showed that the rabbit anti-BPIV3-HNex polyclonal antibody could specifically react with BPIV3 protein.In summary,this study used the prokaryotic expression system to express the BPIV3 HNex protein and obtained rabbit anti-BPIV3 HNex polyclonal antibodies,providing references for further exploration of BPIV3 HN protein function and the development of subunit vaccines.

The aim of this study was to express Feline infectious peritonitis virus (FIPV) N protein by insect cell-baculovirus expression system,and prepare polyclonal antibody against FIPV for clinical antigen/antibody diagnosis and N protein function research.According to the N gene sequence of FIPV in GenBank,the N gene of FIPV strain (accession number:KC461235.1) was selected,and the N gene was optimized by codon optimization and gene synthesis.After enzyme digestion,the N gene was connected to pFastBac1 vector and transformed into E.coli DH5α competent cells.The positive clone was screened by ampicillin resistance.After being identified by restriction enzyme digestion and sequencing,the extracted plasmid was transformed into E.coli DH10Bac competent cells.The recombinant Bacmid-N was successfully constructed after blue and white spot screening and PCR identification.Sf9 cells were transfected with baculovirus and cultured at 28 ℃ for 4 days.The supernatant of Sf9 cells was collected and purified by Ni ion affinity chromatography.The results of SDS-PAGE and Western blotting showed that the recombinant N protein (51 ku) of FIPV was successfully expressed in insect cell-baculovirus expression system.Six weeks old BALB/c mice were immunized with the protein and Freund's adjuvant.The titer of serum antibody was 1:102 400 detected by indirect ELISA.Western blotting and indirect immunofluorescence assay were used to detect and analyze the polyclonal antibody against N protein.The results showed that the recombinant protein expressed in eukaryotic cells had good immunogenicity and the polyclonal antibody had good reactivity,which could specifically identify FIPV infected cells.This study laid a foundation for the development of FIPV antigen/antibody diagnostic kit.

For analyzing the scientific method of using cotton rope to collect oral liquid to monitor pseudorabies (PR) in pig farm,the study was carried out through three aspects,laboratory level,animal test and clinical application.Firstly,the sampling conditions were optimized to determine the ability of the cotton rope to release the Pseudorabies virus (PRV),then the detection time after virus infection was determined by animal experiments,and finally,clinical samples were tested and analyzed.The results showed that it was necessary to suspend a cotton rope with a diameter of 1.0 cm on the pigpen before feeding in the morning,and wait for the pig to bite the cotton rope for 20-30 min.The measurement of virus release ability showed that the release capacity of cotton rope to PRV was about 50%.The measurement of virus release ability showed that 1 TCID₅₀/0.1 mL could be detected.The animal test showed that the pathogenic content in oral fluid was higher than that in nasal swabs except the 5th day in 28 days after infection.And the detection time of the virus (1 d after infection) was earlier than the time when the antibodies turned appear in the blood (7 d after infection).The clinical test results showed that the oral fluid detection method needed to be combined with gE antibody detection to comprehensively determine whether the pig was infected or not,because the vaccine toxicity could also be detected through oral fluids.In summary,this study optimized the oral fluid collection method,and measured the oral fluid release ability of the cotton rope and the detection time after infection,indicating that the oral fluid could be used as a better means of monitoring PR in pigs.In addition,oral fluid detection method needed to be combined with gE antibody detection to comprehensively determine whether the pig herd was an infected group.

The purpose of this experiment was to investigate the growth and decline characteristics of IFN-γ,IL-2,IL-4,IL-10 of sheep after infection with bluetongue virus type 16 (BTV16).In this study,the mRNA of the above four cytokines were detected in three sheep infected with BTV16 using the Real-time quantitative PCR method.At the same time,negative control sheep were set up,and the relative expression of mRNA were calculated based on 0 d mRNA.Meanwhile,virus antibody titers were detected and sheep body temperature was measured.The results showed that all the three sheep inoculated with BTV16 had different degrees of antibody and body temperature symptoms,the mRNA levels of the four cytokines increased significantly within 2-4 days after inoculation of the virus.Among them,the peak value of IFN-γ was 2.58-27.84 times,the peak value of IL-2 was 5.24-17.19 times,the peak value of IL-4 was 2.16-3.43 times,and the peak value of IL-10 was 15.78-48.77 times.There were significant individual differences in the increase amplitude,and all the four cytokines decreased gradually after being kept at a high level for about 6 d.The above parameters of control sheep fluctuated within the normal range with no significant difference (P>0.05).This study elucidated the growth and decline characteristics of IFN-γ,IL-2,IL-4 and IL-10 in sheep inoculated with BTV16 at the transcriptional level,providing references for further studies on the characteristics of BTV infection and the immune mechanism of host organism.

The aim of this study was to obtain highly efficient and specific nanobodies of Bovine viral diarrhea virus (BVDV) NS3 (P80) non-structural proteins.The BVDV inactivated vaccine was used to immunize alpacas and lymphocytes in whole blood were isolated after antibody titer was measured.Phage display technique was used to construct phage display library of heavy chain antibody variable region of alpaca.The phage binding to BVDV-NS3 protein was selected through three successive adsorption-elution-amplification biological screening.Gene sequencing and homology comparison were performed for the cloning of positive nanobody (VHH) identified by PCR and agargel electrophoresis.ELISA was used to verify the reactogenicity of the selected nanobodies,and the nanobodies with high affinity to BVDV-NS3 protein were found.The results showed that the phage display library with insertion rate of 92.8% and storage capacity of 1.84×10¹⁴ CFU/mL was obtained.ELISA results and amino acid sequence analysis showed that a nanometer antibody sequence with good reaction to BVDV-NS3 protein and high homology to VHH was successfully obtained.In this study,Escherichia coli was used to successfully express BVDV-NS3 antigen protein,the phage display library of BVDV nanobody was established,the corresponding nanometer antibodies against the important antigen protein of BVDV were screened and had high homology with VHH.The results provided references for the prevention and control,diagnosis and treatment and the development of nanobody drugs of bovine viral diarrhea-mucosal disease.

This experiment was aimed to estimate the contamination and drug resistance of E.coli in mastitis milk in Shandong.The large-scale dairy farms located in three regions of Shandong province were selected as the sampling sites for this study.A total of 227 milk samples were collected,the bacteriological method was used to isolate and identify E.coli,the micro-broth dilution method was used to detect 11 conventional antibacterial drugs,the 13 common drug resistance genes,8 virulence genes and class Ⅰ integron were screened by PCR.The results showed that a total of 71 E.coli strains were isolated from 227 milk samples.Antibiotic susceptibility testing of E.coli strains showed that strains which were resistant to one or more antibacterial drugs reached 77.5%,the multi-drug resistance rate was 15.5%,of which the resistance rate to polymyxin was 52.2%,and the amoxicillin-clavulanic acid resistance rate was 39.4%.However,all strains were sensitive to neomyci.Of the 71 E.coli strains,all of them carried bla_TEM gene,and they were all bla_TEM-1 gene,32.4% of the strains carried bla_CTX-M gene,and most of them were bla_CTX-M-15 gene.In addition,bla_SHV and bla_OXA genes were not detected;29.6% of the isolates carried mobile colistin resistance genes mcr-1;29.6% of the strains carried aac (6')-Ⅰb-cr gene,and 20.8% carried qnrB gene,qnrA and qnrC genes were not detected.For the analysis of 8 virulence genes,only Hly gene were not detected,and the detection rates of Ecs3703 and Irp2 genes were higher,which were 90.1% and 63.4%,respectively.Class Ⅰ integrons were detected in 11 strains,with a rate of 15.5%,and carried 6 types of gene cassettes,the prevalence of the gene cassette was dfr17-aadA5.The results indicated that the drug resistance of E.coli in mastitis milk in Shandong was serious.E.coli that carrying virulence genes Ecs3703 and Irp2 might be the pathogenic bacteria causing mastitis in dairy cow.The detection of class Ⅰ integrin played a key role in bacterial resistance and gene carrying rate.This study could provide a theoretical basis for clinical prevention and treatment colibacillosis of dairy cow mastitis.

The aim of this study was to investigate the inhibitory effects of antimicrobial peptides Temprine-La(S)(T-La(S)),Temprine-La(FS) (T-La(FS)),RGD-T-La(S) and RGD-T-La(FS) on biofilm formation of Streptococcus suis type 2 (SS2).The biofilm formation ability of SS2 was detected by crystal violet staining (CV).The MBIC and MBEC of antimicrobial peptides against SS2 biofilm were determined by microdilution method.Crystal violet staining and scanning electron microscopy (SEM) were used to detect the effect of antimicrobial peptides on biofilm formation of SS2.XTT method was used to detect the effect of antimicrobial peptides on the biofilm metabolic activity of SS2.Phenol sulfuric acid method was used to detect the effect of antimicrobial peptides on the extracellular polysaccharide content of SS2 biofilm.HE staining was used to observe the effect of T-La(FS) on the pathological changes of zebrafish brain tissue.The effects of antimicrobial peptides on the biofilm related genes of SS2 and the transcription levels of inflammatory cytokines genes in zebrafish were analyzed by Real-time PCR.The results showed that SS2 had good biofilm formation ability.The MBIC of T-La(S),RGD-T-La(S),T-La(FS) and RGD-T-La(FS) on the biofilm of SS2 were 31.3,15.6,7.8 and 15.6 μg/mL,respectively.MBEC were 62.6,31.2,15.6 and 31.2 μg/mL,respectively.The results of crystal violet staining showed that the antibacterial peptides had inhibitory effect on the biofilm formation of SS2.The results of SEM showed that the number of bacteria and the morphology of biofilm of SS2 were significantly changed by antimicrobial peptides,and the extracellular matrix was greatly reduced.The results of XTT showed that antimicrobial peptides could significantly reduce the metabolic activity of the biofilm of SS2.The results of phenol sulfuric acid method showed that antimicrobial peptides could effectively inhibit the synthesis of extracellular polysaccharide from the biofilm of SS2.The results of Real-time PCR showed that the antibacterial peptide reduced the transcription level of biofilm gene of SS2.After treatment with T-La(FS),the transcription levels of TLR2,MyD88 and pro-inflammatory cytokine genes were significantly or extremely significantly decreased (P<0.05;P<0.01),while the transcription levels of anti-inflammatory cytokine genes were significantly or extremely significantly increased (P<0.05;P<0.01).Antimicrobial peptides inhibited the biofilm formation of SS2 mainly by affecting the transcription level of biofilm related genes and blocking the synthesis and secretion of extracellular polysaccharides.T-La (FS) might inhibit the release of inflammatory cytokines and alleviate the inflammatory response of meningitis by inhibiting the expression of TLR2 and MyD88 molecules in TLR2 signaling pathway.

The purpose of this research was to identify the etiology of dead pigeons which clinically suspected Salmonella infection,and to analyze the drug resistance and virulence of pathogenic bacteria.A series of tests including bacterial isolation and culture,colonial morphology observation,staining microscopy,biochemical identification,serotype analysis,16S rRNA gene sequencing analysis,serum plate agglutination test of rehabilitated racing pigeons were used for indentification,drug sensitivity test,drug resistance genes and virulence genes detection were carried out to analyze drug resistance and virulence.The results showed that the isolated bacteria were black colonies on the BS,XLD and HE medium,and the bacteria were Gram-negative short-bacillus without capsule and spores under microscope observation,the biochemical reaction results of the isolated strain conformed to the biochemical characteristics of Salmonella,the serotype of the isolated strain was 1,4,12:i:1,2.The phylogenetic tree analysis of the 16S rRNA gene sequence showed that the isolated bacterial strain and Salmonella Typhimurium were clustered into one branch with the homology >99%.Specific agglutination occurred between the serum of rehabilitated racing pigeons and isolated strain,therefore the isolated bacterial strain was identified as Salmonella Typhimurium considering the results of biochemical identification and serotype detection.The results of drug sensitivity test and drug resistance gene test showed that the isolated bacterial strain was resistant to florfenicol,the floR and cmlA genes of chloramphenicol resistance in isolated bacterial strain were detected which was consistent with the drug resistance phenotype.The isolated bacterial strain was tested positive for 17 virulence genes including mogA and so on.In this study,one strain of Salmonella Typhimurium was successfully isolated and identified from racing pigeons,which was resistant to florfenicol and had highly virulence,providing reference for the prevention and treatment of Salmonella infection in racing pigeons in the next work.

The purpose of this study was to identify the suspected etiology of Rabbit hemorrhagic virus type 2 (RHDV2) infection in a rabbit farm in Jintang,Sichuan province,and to analyze the pathological histological changes of the infected rabbits.Hemagglutination test (HA) and RT-PCR were used to detect and identify the pathogens of the rabbit,and then pathological sections were taken from the diseased tissue to observe and analyze the histopathological changes of each tissue.At the same time,liver suspension was used to infect young rabbits,and the virulence of this strain was analyzed.The results of HA showed that liver samples collected from diseased and dead rabbits could agglutinate human "O" red blood cells.RT-PCR and sequencing results showed that RHDV2 specific bands were amplified from the samples with multiple pairs of primers.The histopathology microscopic examination revealed that the rabbit displayed systemic hemorrhagic and internal swelling with infiltration of large lymphocytes and neutrophils,and the hemorrhagic in liver,lung,and tracheal mucosa were the most serious.The animal test results showed that this strain was highly virulent,and the liver suspension containing RHDV2 was lethal to young rabbits and caused death in 24 h.In this study,clinical diagnosis,nucleic acid testing and sequencing confirmed that the outbreak was caused by RHDV2 infection.Animal tests and histopathological observation showed that this strain was virulent,which could cause serious bleeding organs,resulting in acute death of the sick rabbit.The emergence of RHDV2 indicated that the cross-border spread of virus was not optimistic,and it should be seriously monitored.

The aim of the study was to investigate whether insulin combined with linazolid had both hypoglycemic antibacterial and α-hemolysin activity to inhibit diabetic bacterial pneumonia.The effect of insulin combined with linezolid on macrophage inflammation induced by high glucose and Staphylococcus aureus α-hemolysin was studied in vitro,and its mechanism was discussed.The antibacterial activity of insulin,linezolid alone or in combination with insulin was examined by measuring minimum inhibitory concentration (MIC) in drug sensitivity test and D_{600 nm} to draw growth curve.The inhibitory effect of drug combination on α-hemolysin activity was investigated by hemolysis test.The safety of drug combination in vitro was determined by cytotoxic test.Western blotting was used to analyze the effect of drug combination on inflammatory pathway protein.The results of drug sensitivity test showed that the MIC values of 8325-4 and DU1090 strains of Staphylococcus aureus were not detected in insulin group,and the MIC values of linezolid and insulin+linezolid groups for both strains of Staphylococcus aureus were 0.5 μg/mL.The results of growth curve showed that insulin did not inhibit the growth of Staphylococcus aureus in the normal group and the high glucose group,while in linezolid group and insulin+linezolid groups,0.25 to 4 μg/mL linezolid could inhibit the growth of Staphylococcus aureus.The results of hemolysis test showed that in normal group and high glucose group,insulin did not inhibit α-hemolysin activity,while 0.25 to 4 μg/mL linezolid could inhibit α-hemolysin activity.The cytotoxic test showed that the cell survival rate of normal group and high glucose group was more than 88.038%.The combination of 50 nmol/L insulin,0.25 μg/mL linezolid,50 nmol/L insulin +0.25 μ g/mL linezolid could improve the survival rate of MH-S cells induced by high glucose.The results of Western blotting showed that 50 nmol/L insulin alone did not inhibit the expression of TLR2,MAPKs and NLRP3 in mouse alveolar macrophages (MH-S cells) induced by high glucose and α-hemolysin,while 50 nmol/L insulin combined with 0.25 μg/mL linezolid could reduce the expression level of these proteins in MH-S cells induced by high glucose and α-hemolysin,and the inhibitory effect of linezolid was more significant than that of linezolid alone.In conclusion,insulin combined with linezolid could effectively inhibit the inflammatory response of MH-S cells by lowering high glucose,antibacterial and inhibiting α-hemolysin activity.

This experiment was conducted to study the protective effect of water extract of Rhodomyrtus tomentosa (Ait.) Hass on liver fibrosis rats induced by CCl₄ composited factor,and investigated its mechanism.72 SD mice were randomly divided to normal group,model group,colchicine group,low dose(1.5 mg/g),middle dose (3 mg/g) and high dose (6 mg/g) of water extract of Rhodomyrtus tomentosa (Ait.) Hass,12 mice for each group.Except for the normal group,rats in other groups were subcutaneously injected with 40% CCl₄ peanut oil solution at 1-4 weeks,0.3 mL/100 g body weight,once every other day for 4 weeks.At the same time,30% ethanol was gavaged as 1 mL/piece for 2-4 weeks,once every other day for 3 consecutive weeks.The rats in each group were given gavage at the 5th week,the normal control group and the model group were given normal saline,and the treatment group was given gavage at the corresponding dose,1 mL/100 g body weight,once a day for 4 weeks continuously.The rats in each group were still given gavage at the same time.At the end of the administration,serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity,total bilirubin (TBil) and alpha smooth muscle actin (α-SMA),transforming growth factor β (TGF-β) levels,malondialdehyde (MDA) content and superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) activity were detected.The changes of HE and Masson staining in liver tissues were observed.The results showed that water extract of Rhodomyrtus tomentosa (Ait.) Hass could significantly or extremely significantly reduce the activity/levels of AST,ALT,TBil,TGF-β and α-SMA in rats with liver fibrosis (P<0.05; P<0.01),increase the activity of SOD and GSH-Px in serum (P<0.05),reduce the content of MDA (P<0.05; P<0.01),and reduce the pathological damage degree of liver in rats with liver fibrosis.The above results showed that water extract of Rhodomyrtus tomentosa (Ait.) Hass had an obvious restorative effect on liver function of CCl4 composited factor induced liver fibrosis rats,enhanced its antioxidant capacity,alleviated hepatocyte injury,and had a good effect against liver fibrosis.

Mastitis,which is the most common disease in dairy cows production,can cause inflammation and swelling in the mammary glands of cows,and significantly reduce the milk production.As a multi-factor disease,the reason for mastitis is very complicated.Mastitis is mainly affected by feed contamination,pathogenic bacteria infection,and environmental factors.As the main pathogenic bacteria of mastitis,Streptococcus agalactiae often induces local inflammation of breast tissue.With the increase of the separation rate of Streptococcus agalactiae and the abuse of antibacterial drugs,the resistance of Streptococcus agalactiae to antibacterial drugs gradually increases,which brings certain difficulties to clinical treatment.Additionally,Streptococcus agalactiae can colonize the host,and then damage the immune system of host,and its own virulence factors also can cause disease of host body.With the deepening of relevant researches,it has found that virulence and drug resistance are interrelated with each other.The virulence is affected by the increased drug resistance with different ways,indicating that there is a certain correlation between drug resistance and virulence factors.The authors reviewed research progress of the correlation between Streptococcus and other bacterial virulence genes and drug resistance at home and abroad.The virulence mechanism and virulence genes of Streptococcus agalactiae were analyzed from the perspectives of virulence factors and drug resistance.Besides,the drug resistance of Streptococcus agalactiae in different regions in the past 5 years was recorded.The relationship between Streptococcus virulence and drug resistance was reviewed from the perspectives of bacterial species,environment,and related mechanism,so as to provide theoretical reference for the reasonable selection of antibacterial agents for clinical symptoms induced by Streptococcus agalactiae and relevant researches of virulence genes and drug resistance.

In order to prove the prevention and treatment effect of Chinese herbal medicine compound on broiler bone disease,this experiment selected 160 1-day-old healthy Kosbao broilers and randomly divided into 8 groups to be raised to 30 and 60 days old,a compound of Chinese herbal medicine complex consistion of 8 Chinese herbal medicine such as Angelica sinensis,Fructus Lycii and Dioscorea oppositifolia L..In the experiment,high (0.6%),medium (0.4%),and low (0.2%) dose of Chinese herbal medicine were used,and control group was fed a basic diet.At the age of 30 and 60 days,weight were detected.Biochemical analyzer was used to detect the blood biochemical indexes bone metabolism related (VDR,OT,OPG,ALP),the bone strength (elastic modulus,yield strength and maximum stress) of the tibia was detected by the universal test machine three point bending test,and the Real-time PCR method was used to detect the difference of mRNA expression of OPG,Runx-2,RANK,RANKL,TNF-α and BMP-2 genes in the thoracic and leg cartilage tissue.The results showed that the Chinese herbal medicine compound could gain weight,increase the content of VDR,OT,OPG and ALP in serum (P<0.05) at 60 days old,30 days old was higher than that of 60 days old.It could enhance tibial bone strength (P<0.05),30 days old was higher than that of 60 days old.And it could also promote the expression of OPG,BMP-2 and Runx-2 genes and inhibit the expression of TNF-α,RANK and RANKL genes (P<0.05).From above mentioned results,considering the relative indexes of live weight and bone strength,compound Chinese herbal medicine high dose group was the best formula to improve the bone characteristics of the broilers.The compound Chinese herbal medicine could improve the content of VDR,OT,OPG and ALP in serum,regulate the OPG/RANK/RANKL system to maintain the dynamic balance of bone resorption and bone formation,promote the expression of BMP-2 and Runx-2 genes in the important pathway of bone formation BMP-2/Smads/Runx-2,and inhibit TNF-α gene expression,so as to improve the skeleton characteristics of Kosbao broilers,and effectively prevent bone disease of broilers.

At present,milk has become an important part of people's dietary structure.It can not only provide rich nutrition,but also has certain health functions.The function mining and evaluation of milk nutrition is a research hotspot in the field of food nutrition.The function of milk nutrition mainly depends on the active factors,including active protein/polypeptide,active fatty acid,etc.The author reviewed and summarized the literature on the functional evaluation of active factors in milk in recent years,and extracted and summarized several important active proteins from them,mainly including lactoferrin,α-lactalbumin and β-lactoglobulin.The anti-inflammatory,antibacterial,antiviral,anti-tumor and immunomodulatory functions of these three active proteins were systematically explained.At the same time,we also studied several important active fatty acids in milk,including oleic acid,linolenic acid and docosahexaenoic acid,and elaborated their anti-inflammatory,antihypertensive,cardiovascular and cerebrovascular disease related activities and mechanisms.For the specific elaboration of active protein and active fatty acid in milk,the relevant experimental investigation was taken as the breakthrough point,and the mechanism was that the active factors directly or indirectly acted on the target cells and played related functions.