【Objective】 This study was aimed to analyze the bioinformatics properties of two Kunitz-type serine protease inhibitor genes (EGR-07243 and EGR-07244) of Echinococcus granulosus,and perform the prokaryotic expression,so as to detect the immunogenicity of EGR-07243 and EGR-07244 proteins.【Method】 The full-length sequences of EGR-07243 and EGR-07244 genes were amplified by PCR,the physicochemical properties,structural characteristics and phylogenetic relationship of EGR-07243 and EGR-07244 proteins were analyzed by bioinformatics software.The recombinant plasmids pET32a-EGR-07243 and pET32a-EGR-07244 were constructed and transformed into E.coli BL21 (DE3) competent cells.The protein expression was detected by SDS-PAGE,and the antigenicity of the two proteins was analyzed by Western blotting.【Result】 The full-length of EGR-07243 gene was 228 bp,encoding 75 amino acids, the protein molecular formula was C₆₈₆H₁₁₄₆N₂₂₈O₂₉₂S₆₃,the relative molecular mass was about 19 258,containing 3 B cell antigen epitopes,the theoretical isoelectric point was 5.30,the fat coefficient was 17.98,and the hydrophilicity was 0.714,which was a stable protein.The full-length of EGR-07244 gene was 228 bp,encoding 75 amino acids,the protein molecular formula was C₆₇₅H₁₁₂₄N₂₂₈O₂₈₇S₅₆,the relative molecular mass was about 18 800,containing 3 B cell antigen epitopes,the theoretical isoelectric point was 5.32,the fat coefficient was 20.61,and the hydrophilicity was 0.689,which was a stable protein.The amino acid sequence similarity of the two proteins was 72.00%.The amino acid sequence identity of the two domains with Taenia genus were more than 95%,which were closely related.However,and were 46.3% with human,which were far related.Double enzyme digestion and sequencing analysis showed that the recombinant plasmids pET32a-EGR-07243 and pET32a-EGR-07244 were successfully constructed.The recombinant plasmids were transformed into E.coli BL21 (DE3) competent cells,and the expression proteins were successfully induced.Western blotting results showed that the two proteins could be recognized by positive sera of dogs infected with protozoa but not by sera from healthy dogs,indicating that the two proteins had good reactivity.【Conclusion】 EGR-07243 and EGR-07244 proteins could be used as vaccine immune targets in the prevention of fine-grained Echinococcus granulosus infection.The results provided experimental materials for screening candidate molecules of anti-echinococcosis vaccine.

【Objective】 The aim of this study was to screen long non-coding RNA (lncRNA) that might had a "sponge adsorption" effect on bta-miR-146a targeting Smad family member 4 (Smad4) gene during follicle development of yak.【Method】 The miRanda and RNAhybrid databases were used to predict lncRNA targeting bta-miR-146a in yak.The ovary in yak was used to isolate healthy and atretic follicles,and the total RNA was extracted by Trizol method and reverse transcribed into cDNA.The expression of predicted lncRNA was detected by PCR,and the expression of lncRNA and bta-miR-146a/Smad4 in healthy and atretic follicles of yak were detected by Real-time quantitative PCR.The wild-type and mutant dual luciferase vectors of lncRNA-ENSBGRT00000000387.1 were constructed and cotransfected with bta-miR-146a mimics and mimics NC into HEK293T cells to detect the double luciferase activity.【Result】 The results showed that 7 lncRNAs were predicted to have "sponge adsorption" effect on bta-miR-146a targeting Smad4 gene,and the expression of lncRNA-ENSBGRT00000000387.1 in follicles of yak was extremely significantly higher than other lncRNAs (P＜0.01).Real-time quantitative PCR results showed that lncRNA-ENSBGRT00000000387.1,bta-miR-146a and Smad4 gene mRNA were co-expressed in healthy and atretic follicles of yak,and the expression of lncRNA-ENSBGRT00000000387.1 and Smad4 gene mRNA in healthy follicles of yak were significantly higher than that in atretic follicles (P＜0.05),but the expression of bta-miR-146a was extremely significantly lower in healthy follicles of yak than that in atretic follicles (P＜0.01).lncRNA-ENSBGRT00000000387.1 wild-type and mutant pmirGLO dual luciferase reporter plasmids of yak were successfully constructed,and the dual luciferase activity results indicated that bta-miR-146a mimics had an extremely significant down-regulation effect on lncRNA-ENSBGRT00000000387.1-WT of yak (P＜0.01).【Conclusion】lncRNA-ENSBGRT00000000387.1,bta-miR-146a and Smad4 gene mRNA might have regulatory mechanisms in follicle development or atresia process of yak,and it was tentatively confirmed in vitro that lncRNA-ENSBGRT00000000387.1 and bta-miR-146a had "sponge adsorption" effect,which provided the reference for further study of the functional mechanism of lncRNA-ENSBGRT00000000387.1 in follicles of yak.

【Objective】 The purpose of this study was to amplify the full-length coding sequence (CDS) of cryptochrome circadian regulator 1 (CRY1) gene in dairy cows,construct its eukaryotic expression vector and conduct bioinformatics analysis,and detect the expression profiles of CRY1 gene in dairy cows,so as to provide a reference for subsequent exploration of the biological function of CRY1 protein.【Method】 The CDS region of CRY1 gene was amplified by PCR using the cDNA template from liver in dairy cows.The CDS of CRY1 gene was connected to the enzyme digesting linearized pcDNA3.1-3HA empty plasmid by homologous recombination method.The recombinant plasmid with correct sequence was named pcDNA3.1-3HA-cCRY1.Bioinformatics software was used to analyze CRY1 gene in dairy cows.The pcDNA3.1-3HA empty plasmid and pcDNA3.1-3HA-cCRY1 recombinant plasmid were transfected into HEK293T cells,respectively.Western blotting was used to detect the expression of CRY1 protein.Real-time quantitative PCR was used to detect the expression profile of CRY1 gene in different tissues of dairy cows.【Result】 The CDS region (1 764 bp) of CRY1 gene was successfully obtained,and the eukaryotic expression vector pcDNA3.1-3HA-cCRY1 was successfully constructed.The CDS region of CRY1 gene in dairy cows was more than 98% similarity to Bos mutus,Capra hircus and Ovis aries,and their genetic distance were close relatively.Bioinformatics analysis showed that CRY1 protein was alkaline and hydrophilic protein,without transmembrane domain and signal peptide.CRY1 protein had 45 potential phosphorylation sites,and interacted with CLOCK,ARNTL,FBXL3 and other proteins.Western blotting result showed that the pcDNA 3.1-3HA-cCRY1 transfection group had a distinct band at 72 ku,while the pcDNA 3.1-3HA transfection group had no band at the corresponding locations.Real-time quantitative PCR results showed that CRY1 gene in dairy cows was expressed in 10 tissues such as heart,liver,spleen and lung,with the highest expression in heart and the lowest expression in trapezius cervical muscle.【Conclusion】 In this study,the eukaryotic expression vector of CRY1 gene was successfully constructed,and the overexpression of CRY1 protein was achieved in HEK293T cells.CRY1 protein was an unstable intracellular protein that could interact with multiple proteins and participate in circadian rhythm regulation,cell metabolism,gluconeogenesis and other processes. CRY1 gene was conserved among ruminants such as Bos taurus and Ovis aries,which was distributed in various tissues of dairy cows.The results provided a reference for further exploration of the transcriptional regulation mechanism of CRY1 protein in the biological clock system of dairy cows.

【Objective】 The purpose of this study was to investigate the biological evolutionary process and potential target gene function of miR-192,and the expression differences of miR-192 in adipose tissue of Daweizi pigs (obese-type) and Yorkshire (lean-type).【Method】 miR-192 mature sequences were obtained using the miBase database,and the distribution characteristics of miR-192 on the genomes of different species was located using the Ensembl database.The phylogenetic tree of miR-192 was constructed using the Mega 11.0. The expression of miR-192 in the dorsal adipose tissue of Daweizi pigs and Yorkshire were analysised by quantitative Real-time PCR,and the expression differences were compared.Multiple target gene prediction sites were used to predict the target genes that might bind to pig miR-192,and GO function and KEGG enrichment analysis were performed on the target genes.【Result】 miR-192 maintained a high degree of conservation during biological evolution.Quantitative Real-time PCR results showed that the expression of miR-192 in the dorsal adipose tissue of Daweizi pig was extremely significantly higher than that of Yorkshire (P<0.01). The results of GO function and KEGG pathway enrichment showed that the target gene of miR-192 mainly play the role of pre-transcriptional regulation and binding,and mainly function in chromatin and nucleus,and most genes were enriched in signaling pathways related to adipose metabolism.Five conserved target genes of pig miR-192 were obtained from the intersection of prediction results of different species on three websites.【Conclusion】 miR-192 was highly conserved in biological evolution and might regulate the expression of target genes by targeting,and played an important role in adipose tissue differentiation of pigs,which provided a theoretical basis for further research on the regulatory mechanism of miR-192 in adipose tissue differentiation.

【Objective】 The purpose of this study was to analyze the structural characteristics of the promoter region sequence of MyoG gene and its transcriptional regulation mechanism.【Method】 The blood genomic DNA of Guizhou White goat was used as template,two pairs of primers were designed,and the promoter region of MyoG gene was obtained by PCR amplification and Sanger sequencing.The sequence similarity comparison and phylogenetic tree construction of MyoG gene promoter region among different species were carried out by DNAStar and Mega 5.0 software.The core promoter region,transcription factor binding site,cis-acting element and CpG island of obtained sequence were predicted and analyzed by bioinformatics software.【Result】 The length of promoter region of MyoG gene in Guizhou White goat was 2 334 bp,including the 5'-flanking region of 2 092 bp and 242 bp of exon 1,and the GC content was slightly lower than that of AT.Through the comparative analysis of different species,the similarity of the promoter region of MyoG gene in Guizhou White goat with that of Ovis aries,Bos taurus,Sus scrofa,Homo sapiens,Mus musculus,and Gallus gallus were 98.34%,96.15%,77.76%,74.15%,57.63% and 43.04%,respectively.Phylogenetic tree revealed that Guizhou White goat was the most closely related to Ovis aries and Bos taurus,and the farest related was Gallus gallus.Bioinformatics structure prediction found that the transcription initiation site of MyoG gene in Guizhou White goat was located at 50 bp upstream of the translation initiation codon ATG,and there was a potential promoter region and a CpG island in the 5'-flanking region,containing 1 TATA-box (TTTAAATG),1 GC-box(CCGCCC),2 CAAT-box(CCAAT),17 E-box(CAGATG) and 1 A/T rich element (TATATTTAT). The potential binding sites of Sp1,NF-1,MEF2,MyoD,USF,GATA-1,GATA-3,SRF,C/EBP and c-Myb transcription factors were predicted by AliBaba 2.1,PROMO,Patch 1.0,JASPAR²⁰²² and AnimalTFDB 3.0 online software.【Conclusion】 The promoter region sequence of MyoG gene in Guizhou White goat was successfully cloned,which provided a theoretical basis for further research on the molecular mechanism of MyoG gene regulating goat muscle development.

【Objective】 The purpose of this experiment was to study the lipid profile of duck breast muscle and investigate the differences between the breast muscle lipidome of Pekin duck and Liancheng White duck.【Method】 28 Pekin ducks and 28 Liancheng White ducks were selected at 6-week-old and their breast muscle tissues were collected to detect the lipid profile used lipidomics techniques based on liquid chromatography-mass spectrometry (LC-MS).The results were analyzed by multivariate statistical analysis,and the differential lipids were screened by fold change,t-test and partial least squares discriminant analysis (PLS-DA).【Result】 A total of 950 lipids were identified in duck breast muscle,covering 6 categories,395 glycerophospholipids (GP),365 glycerolipids (GL),86 sphingolipids (SP),78 fatty acyls (FA),23 sterol lipids (ST) and 3 prenol lipids (PR);The most abundant lipid subgroup was triglycerides (TG) with 283 species.Compared with Pekin duck,18 lipids were screened in Liancheng White duck, 7 were upregulated and 11 were downregulated;Among them,TGs were the most abundant and high in Pekin duck,which were rich in saturated and monounsaturated fatty acids,while these GP containing very long chain polyunsaturated fatty acid (VLC-PUFA) were found to be high in Liancheng White duck.【Conclusion】 In this study,a comprehensive and detailed lipid profile of duck breast muscle was obtained,and 18 differential lipid markers were screened for Pekin duck and Liancheng White duck,this study provided a scientific and theoretical basis for intramuscular lipid metabolism,meat quality research and quality breeding of duck.

【Objective】 The purpose of this experiment was to analyze the effects of Guiqi Yimu oral liquid on plasma metabolites in postpartum dairy cows by UPLC-MS/MS metabolomics technology,in order to clarify the health-care mechanism of Guiqi Yimu oral liquid on postpartum dairy cows.【Method】 A total of 15 healthy Holstein dairy cows with similar body condition and 2-3 parity were selected and randomly divided into control group (n=7) and experiment group (n=8).500 mL/head Guiqi Yimu oral liquid and drinking water were administered continuously for 6 days.Blood was collected before morning feeding on 7 d postpartum.After pretreatment,UPLC-MS/MS was used for on-machine detection,and MetaboAnanlyst 5.0 software was used to screen differential metabolites with VIP≥1 and P＜0.05 for metabolic pathway enrichment analysis.【Result】 At 7 days postpartum,the plasma metabolism profile in experiment group was significantly changed compared with control group.Sixteen differential metabolites were screened out,of which eight were significantly up-regulated,including methionine sulfoxide,creatine phosphate,glycerophosphocholine,cortisol,L-tryptophan,lysophosphatidylcholine(18∶2),5-hydroxytryptamine and L-arginine;Eight were significantly down-regulated,including 1-aminocyclohexanoic acid,3-methylcrotonyl glycine,L-asparagine anhydrous,monoacylglycerolacyl(16∶1),panthenyl mercaptoethylamine,octadecy monoenamide,1-methylguanine and 3-N-methyl-L-histidine.Differential metabolites were significantly enriched in glycerophospholipid metabolism,arginine and proline metabolism,and tryptophan metabolism pathways.【Conclusion】 Guiqi Yimu oral liquid had the health-care effect of postpartum dairy cows,which might play the role of anti-inflammatory,antioxidant,appetite-regulating,intestinal bacteria and relieving negative energy balance by regulating glycerophospholipid metabolism,cortisol metabolism,tryptophan metabolism,arginine and proline metabolism.

Bile acids not only contribute to digestion and nutrient absorption,but also have recently been shown to be a signaling molecule involved in the regulation of multiple physiological functions through the activation of corresponding receptors,such as lipid metabolism,glucose metabolism and energy metabolism.The gut-brain axis is a two-way signal system composed of the gastrointestinal tract and the central nervous system,which not only integrates the functions of the gut and the central nervous system,but also organically connects them.Bile acids interact with the gut to some extent,and the gut microbiota is the key factor of the interaction between bile acids and the gut,and also the important factor of the gut-brain axis.The effects of bile acids in different concentrations on the intestinal tract are different.The effects of bile acids on the intestinal tract are mainly reflected in their effects on the intestinal microbes,a large part of the effect of the gut on bile acids is also due to gut microbes.At present,many researches focus on the interaction between bile acids and gut microbes.In addition,bile acids and bile acid receptors have also been found in the brain,suggesting that bile acids may play a physiological role in central nervous system.Bile acids can directly or indirectly signal the central nervous system to affect various brain functions.A variety of gut hormones,vagus nerves and bile acid receptors are involved in this process,among which bile acid receptors play an important role.More and more studies show that there are complex interactions between bile acids,brain,gut and gut microbes.Bile acids may be one of the direct communication channels between gut and brain,and have potential effects on gut-brain axis.In this paper,the effects of bile acids and their receptors in the gut and brain were reviewed,and the research progress of bile acids in the gut-brain axis was introduced.

【Objective】 The aim of this study was to investigate the effects of spontaneous movement,exploratory behavior,anxiety and blood hormone indexes in ICR mice with different cold stress rewarming.【Method】 Compared with 28 ℃ normal temperature control group,the movement changes of ICR mice in the open field test and elevated plus maze test after rewarming 2,4,6,8,10 and 12 h in 4,10,16 and 22 ℃ cold stress rewarming group were detected,respectively,and the changes of blood hormone indexes in each group after rewarming 2 and 4 h were detected by ELISA.【Result】 The results of open field test showed that compared with 28 ℃ normal temperature control group,the residence time and exercise time of central region were significantly reduced after 10 h of rewarming at 22 ℃ cold stimulation (P＜0.05).The residence time in the central region was significantly decreased after 4 and 8 h of rewarming at 16 ℃ cold stimulation (P＜0.05).The residence time in the central region was significantly decreased after 4 and 10 h of rewarming at 10 ℃ cold stimulation (P＜0.05),and the movement distance of the central region was significantly increased after 2 h of rewarming (P＜0.05).The residence time of the central region was extremely significantly decreased after 6 h of rewarming at 4 ℃ cold stimulation (P＜0.01),and the movement distance of the central region was extremely significantly increased after 2 h of rewarming (P＜0.01).Elevated plus maze test showed that compared with 28 ℃ normal temperature control group,the residence time of closed arms was significantly or extremely significantly decreased after 4 h of rewarming at 22 and 4 ℃ cold stimulation and 10 h of rewarming at 16 ℃ cold stimulation (P＜0.05 or P＜0.01).There was no significant change in the residence time of closed arm in 10 ℃ cold stimulation rewarming group (P＞0.05).Corticosterone (CORT) was extremely significantly increased after 4 h of rewarming at 22 ℃ cold stimulation (P＜0.01),and was extremely significantly increased after 2 and 4 h of rewarming at 16 ℃ cold stimulation (P＜0.01).Epinephrine (EPI) was extremely significantly increased after 4 h of rewarming at 22 ℃ cold stimulation (P＜0.01),and extremely significantly increased after 2 and 4 h of rewarming at 10 ℃cold stimulation (P＜0.01).【Conclusion】 After the end of cold stress,the spontaneous movement,exploration behavior,anxiety and blood hormone indexes of ICR mice would continue to be affected.It was necessary to reduce the temperature change of the test environment with ICR mice as the model and improve the accuracy of the test results.

Lysozyme is a natural antibacterial agent,which could directly hydrolyze the bacterial cell wall to generate antibacterial activity,however,the property that lysozyme only has antibacterial activity against Gram-positive bacteria limits its application in the field of food and breeding.Lysozyme is a protein with small molecule,more and more studies have modified lysozyme to improve its antibacterial activity and expand its antibacterial spectrum.This paper mainly reviewed that the antibacterial activity of lysozyme had been greatly improved after modification using high temperature,pH,metal ions,chemical modification,combination with nanomaterials or other solutions,and genetic engineering technology,especially for Gram-negative bacteria.The bacteriostatic activity of lysozyme can be improved by the following ways:①Destroying the non-covalent binding within the enzyme molecule by raising the temperature;②Affecting the molecular space structure of the enzyme by reducing pH;③Adding metal ions;④Chemical modification of side chain group or chemical group of enzyme molecule;⑤Lysozyme is loaded on nano materials and fixed into nanocomposites to destroy cell walls;⑥The lysozyme and other solutions inhibiting the growth of bacteria are mixed to obtain a composite solution;⑦Construction of new lysozyme expression vector using gene recombination technology.The research and transformation of lysozyme has important guiding significance for the development of basic science,the application of lysozyme in aquaculture and livestock breeding instead of antibiotics,and the development of green natural food preservatives.

【Objective】 The purpose of this experiment was to investigate the effect of yeast selenium supplementation on the gastrointestinal microflora of sheep under grazing conditions.【Method】 Fourteen healthy 7-8 months old Dolphin sheep with similar body weight were selected and divided into two groups by single factor complete randomized trial design,with seven sheep in each group.One group was the control group without adding selenium,and the other group was the selenium adding group.The selenium (yeast selenium) adding level was 0.2 mg/d.A 30 day feeding experiment was conducted under natural grazing conditions.Rumen juice and feces samples were collected on the 15th and 30th days of the experiment,respectively.The 16S rRNA sequencing method was used to analyze the changes of rumen and hindgut microflora of sheep.【Result】 The results of principle coordinate analysis showed that rumen and hindgut microbes of sheep in two groups had significant differences in OTU and genus levels after 30 days of selenium supplementation (P＜0.05).The results of discriminant analysis of species differences showed that eight rumen microorganisms had significant differences between groups at phylum and genus levels on the 15th day of feeding (P＜0.05),but the abundance of these species was lower than 1%. At the 30th day of feeding,there were 17 rumen microorganisms with significant differences between groups at the phylum and genus levels (P＜0.05),among which Firmicutes,Bacteroidetes,Prevotella-1,Ruminococcaceae_NK4A214 and Bacteroidales_BS11_gut_group had relative abundance greater than 1%.Compared with the control group,the relative abundance of Firmicutes,Ruminococcaceae_NK4A214 and Bacteroidales_BS11_gut_group in the rumen in selenium supplementation group was significantly increased (P＜0.05),while the relative abundances of Bacteroidetes and Prevotella_1 was significantly decreased (P＜0.05).At the genus level,on the 15th day,there were significant differences in five species of posterior intestinal microorganisms between groups (P＜0.05),but the abundance of these species was lower than 1%.On the 30th day,there were 18 species of posterior intestinal microorganisms with significant differences between groups at phylum and genus level (P＜0.05),among them,the relative abundance of Firmicutes,Bacteroidetes,Bacteroidetes,Ruminococcaceae_UCG-009,Ruminococcaceae_UCG-010,Ruminococcaceae_UCG-013 and Ruminococcaceae_NK4A214,norank_f_Ruminococcaceae,Lachnospiraceae_AC2044 and norank_f_Muribaculaceae were greater than 1%.Compared with the control group,the relative abundances of Bacteroidetes,Bacteroidetes,Ruminococcaceae_UCG-010 and Ruminococcaceae_UCG-013 in intestinal tract of selenium supplementation group were significantly increased (P＜0.05).The relative abundances of Firmicutes,Lachnospiraceae_AC2044,Ruminococcaceae_NK4A214 and Ruminococcaceae_UCG-009,norank_f_Ruminococcaceae and norank_f_Muribaculaceae were significantly decreased (P＜0.05).【Conclusion】 Selenium supplementation of sheep under grazing conditions could improve rumen microbial diversity and hindgut microbial richness,increase the abundance of fiber-degraded bacteria in rumen as well as protein polysaccharide-degraded bacteria in hindgut.In addition,selenium supplementation could promote probiotics growth and inhibited the platation of harmful microorganisms in hindgut of sheep.

【Objective】 This experiment was designed to investigate the appropriate dietary comprehensive net energy requirement (NEmf) for Angus bulls at the late stage of continuous fattening.【Method】 A factor complete randomized trial design was adopted.30 Angus bulls with similar monthly age (18.3±1.0) and body weight ((503.3±21.1) kg) were randomly divided into 3 groups,namely low energy group (LE group),medium energy group (ME group) and high energy group (HE group),with 10 cows in each group and one cow per replicate.The experimental period was divided into two stages:The early stage (500-600 kg) and the late stage (600-700 kg),and the comprehensive net energy of the diets was 6.64,6.84,and 7.04 MJ/kg in the early stage and 6.84,7.04,and 7.24 MJ/kg in the late stage,respectively,and the protein level of the diets was 11.42% in two stages.The test period was 10 d for the pre-feeding and 130 d for the trial,and the growth performance and body size indexes of Angus bulls were weighed before the morning feeding and blood samples were collected from the tail root vein to determine the serum biochemical indexes at the beginning and end of each trial.The economic benefits were calculated based on feed cost and weight gain. 【Result】 ①In the early and late stages of the experiment,the total weight gain and average daily weight gain of Angus bulls in medium and high energy groups were significantly higher than those in low energy group (P＜0.05),and the feed-to-gain ratio was significantly lower than that in low energy group (P＜0.05).②In the early stage of the experiment,the serum triglyceride content of high-energy group was significantly higher than that of low-energy group (P＜0.05),and the dietary energy level did not significantly affect the final weight and average dry matter intake (P＞0.05).The economic efficiency was best in high-energy group.③In the late stage of the experiment,the final body weight of medium and high energy groups was significantly higher than that of low energy group (P＜0.05).The average dry matter intake,chest circumference and abdominal circumference of high energy group were significantly higher than that of low energy group (P＜0.05).The economic efficiency was best in medium energy group.④Live weight before slaughter and perirenal fat weight were significantly higher in medium and high-energy groups than that in low-energy group (P＜0.05).Carcass weight and backfat thickness were significantly higher in high-energy group than that in low-energy group (P＜0.05).⑤Dietary energy level had no significant effect on serum glucose and urea nitrogen levels,slaughter rate and eye muscle area (P＞0.05).【Conclusion】 Appropriate increase of dietary energy level could maintain the normal energy metabolism of the body and improve the fattening efficiency of Angus bulls.Comprehensive analysis showed that the appropriate dietary energy (comprehensive net energy) level for Angus bulls in the late stage of continuous fattening was 7.04 MJ/kg.

Transportation is an important part during poultry production to slaughter.With the increasing demand for poultry products from consumers,poultry breeding industry has been developing rapidly,which makes transportation become more and more frequent in poultry.The impact of poultry transportation has attracted much attention.Transportation stress refers to the combined effects of various stressors,such as hunger,thirst,climate,crowding,noise,vibration,mixing,physical consumption,transport duration and pathogen invasion,which cause transport stress syndrome.Transportation stress would lead to the decline of poultry production performance or even death,cause the abnormal function of neuroendocrine,immune and energy supply systems in poultry,cause damage to the body tissues,worsen the meat quality,and also induce intestinal oxidative stress and lead to gut microbiota disorder,damage intestinal health,and thus affecting the growth,development and health of poultry.These not only damage poultry welfare,but also bring serious economic losses and resource waste to the poultry industry.Therefore,the transportation stress of poultry could be alleviated by shortening the transportation time,giving appropriate pre-slaughter rest,controlling density,preventing cold and cooling,and supplementing Chinese herbal medicine and plant extracts,trace elements and other functional additives.The authors summarized the effects of transportation stress on poultry production performance,endocrine and immune performance,meat quality and intestinal health,and reviewed the related measures to alleviate transportation stress,in order to provide important reference for research on poultry transportation stress and its mitigation regulation.

【Objective】 This experiment was conducted to investigate the effects of Scutellaria baicalensis georgi(SBG) on antioxidant capacity,sex hormone levels and mRNA expression of estrogen receptors α(Erα) and progesterone receptors(PR) in female mice exposed to zearalenone(ZEA).【Method】 Fifty female mice were chosen and randomly divided into five groups after one week pre-feed,with 10 mice in each group.The mice in control group (CK) were given 10 mL/kg corn oil,in ZEA group were given 20 mg/kg ZEA,in ZEA+SBG10 group were given 20 mg/kg ZEA+10 g/kg SBG,in ZEA+SBG20 group were given 20 mg/kg ZEA+20 g/kg SBG, and the mice in ZEA+SBG30 group were given 20 mg/kg ZEA+30 g/kg SBG.All experimental mice were fed a basal diet for 3 weeks.The body weight of mice was recorded daily during the trial period.The blood samples of tail root vein of mice were collected before morning feeding at the end of the first,second and third week,respectively,and the antioxidant-related indexes and sex hormone content in serum were determined.At the end of the third week of the trial,ovaries and uterus were collected to detect the mRNA expression of the relevant steroid hormone receptor gene.【Result】 Compared with the CK group,ZEA exposure for more than 2 weeks resulted in a significant decrease in body weight,serum glutathione peroxidase (GSH-Px) activity,glutathione (GSH) and estradiol (E₂) levels (P＜0.05),and a significant increase in ovarian stimulating hormone (FSH) levels (P＜0.05). While luteinizing hormone (LH) levels had no significant changes (P＞0.05).Compared with ZEA group,the body weight of mice treated with 20 or 30 g/kg SBG for 3 weeks was significantly increased (P＜0.05),and the activities of total superoxide dismutase(T-SOD) and GSH-Px and GSH level in serum of mice treated with 20 g/kg SBG for 3 weeks were significantly increased (P＜0.05).After treatment with 30 g/kg SBG for 2 weeks,these three indexes were significantly increased (P＜0.05).Treatment with 20 or 30 g/kg SBG for 2 weeks significantly decreased serum malondialdehyde(MDA) content (P＜0.05).Treatment with 30 g/kg SBG for 2 weeks significantly increased the level of E₂ (P＜0.05),and significantly decreased the content of FSH (P＜0.05).After 3 weeks of treatment with 20 or 30 g/kg SBG,ERα gene expression in uterus and ovary was significantly increased (P＜0.05),but there was no significant difference in PR mRNA expression among all groups (P＞0.05).【Conclusion】 SBG could improve the serum antioxidant capacity and sex hormone levels of female mice exposed to ZEA,enhance the expression of uterine and ovarian ERα,and alleviate the oxidative damage and reproductive toxicity caused by ZEA. The recommended dose of SBG was 20 to 30 g/kg.

【Objective】 The purpose of this experiment was to explore the effects of adding different concentrations of coated sodium butyrate in health sand on the growth performance,slaughter performance and immune organ index of breeding pigeons and squabs.【Method】 A total of 336 pigeons at 6 months of age were randomly divided into 4 groups with 7 replicates per group and 12 in each replicate.Squabs were natural hatching by breeding pigeons,each pair of breeding pigeon fed two squabs.The breeding pigeons in the control group were fed a basal health sand,the breeding pigeons in groups A to C were fed basal health sand supplemented with 0.1%,0.2% and 0.4% coated sodium butyrate.The experiment of breeding pigeons lasted for 120 days and that of squabs lasted for 28 days.The initial and final weights of breeding pigeons and squabs were weighed, the feed consumption of breeding pigeons was counted,and the growth performance was calculated.At the end of the test,5 breeding pigeons and 5 squabs in each group were selected and slaughtered,and then the slaughter weight,breast muscle weight,leg muscle weight,semi-eviscerated weight and eviscerated weight were weighed,and the slaughter performance was calculated.The thymus,spleen,bursa of Fabricius of squabs and thymus and spleen of breeding pigeons were weighed to calculate the immune organ index.The activities of amylase,trypsin and lipase in duodenum of breeding pigeons were determined.【Result】 ①The average daily weight gain of breeding pigeons in groups A and B during lactation was significantly higher than that of the control group and group C,and the feed intake of breeding pigeons aged 10 months in group A was significantly higher than that of the control group (P＜0.05),while the average daily weight gain of squabs in groups B and C was significantly higher than that of the control group and group A (P＜0.05).②Compared with the control group,the percentage of semi-eviscerated yield and eviscerated yield of breeding pigeons in groups A and B,and the percentage of semi-eviscerated yield and eviscerated yield,slaughter rate of squabs in groups A,B and C were significantly increased (P＜0.05).③There was no significant difference in thymus index and spleen index of breeding pigeons among the groups (P＞0.05).The thymus index of squabs in group B was significantly higher than that in group C,while the bursal of Fabricius index of squabs in group B was significantly lower than that in group C (P＜0.05).④Compared with the control group,the trypsin activity in the duodenum of breeding pigeons in group C was significantly higher (P＜0.05),and the lipase activity in groups B and C was significantly higher (P＜0.05).【Conclusion】 Adding coated sodium butyrate to healthy sand could improve the average daily gain and average feed intake of breeding pigeons and squabs,improve slaughter performance and immunity of squabs,and increase trypsin activity and lipase activity in duodenum of breeding pigeons.Under the experimental conditions,adding 0.2% coated sodium butyrate in the health sand had the best effect.

【Objective】 This study was aimed to study the protective effect of honokiol (HNK) on renal injury in animals exposed to cadmium (Cd).【Method】 Forty eight Sanhuang chickens with similar body weight (61.05 g±2.00 g) and good health conditions were randomly divided into four groups,each group had three replicates and each replicate had four chickens.The chickens in control group was fed with basal diet,in Cd group, 70 mg/kg cadmium chloride were added in the basal diet,in HNK group, 400 mg/kg HNK were added in the basal diet, and in the combined treatment group (HNK+Cd group), 70 mg/kg cadmium chloride and 400 mg/kg HNK were added in the basal diet.The feeding period was 30 days.【Result】 Compared with control group,the content of cadmium in the kidney,the serum uric acid (UA) and creatinine (CREA) contents were extremely significantly increased (P＜0.01),the lumen of the renal crypt was enlarged and the number of apoptotic cells was increased,the contents of selenium,iron,and manganese in kidney were extremely significantly decreased (P＜0.01),and the contents of zinc and copper in kidney were extremely significantly increased(P＜0.01) in Cd group.The contents of zinc and iron in kidney were extremely significant or significantly increased (P＜0.01 or P＜0.05),and there were no significant differences in the cadmium,selenium,copper,and manganese contents of kidney,and serum UA and CREA contents in HNK group (P＞0.05).Compared with Cd group,for the chickens in HNK+Cd group,the contents of cadmium,copper in kidney and serum UA were significantly decreased (P＜0.05),the content of CREA was decreased,but the difference was not significant (P＞0.05),the contents of selenium,zinc,iron,and manganese in kidney were significantly increased (P＜0.05),and the renal tubular cell injury was alleviated and the number of apoptotic cells was also reduced.【Conclusion】 HNK could reduce the accumulation of cadmium in kidney,reduce the number of apoptotic cells in kidney tissue,alleviate renal injury,improve the metabolism disorder of trace elements in chicken,and enhance the constitution of chicken.

Probiotics are a kind of microorganisms that contribute to the health of animals.They play an important role in improving the immunity of animals,the balance of intestinal microorganisms and the environment of livestock farms.In recent years,scholars in domestic and foreign have gradually deepened their exploration of genetic engineering technology which applied it in practical production.However,the vast majority of research has focused on eukaryotes.On this basis,how to apply gene editing technology to probiotics and make probiotics play a greater potential is the current research upsurge.Therefore,through genetic engineering technology,specific genes and probiotics can be gene edited and expressed,and the edited probiotics can express specific genes or target to play an immunomodulatory role on the host.In this paper,the interaction between probiotics gene-edited and host have been summarized.The application of CRISPR-Cas9 technology in gene editing lactic acid bacteria and animal production is reviewed.Meanwhile,the methods of gene editing of lactic acid bacteria by different gene editing technologies are analyzed and compared.It is found that CRISPR-Cas9 technology is a relatively flexible gene editing tool for food-grade probiotics such as lactobacillus and bifidobacteria,so as to realize the purpose that probiotics can play multiple health effects in the host body,and the future application of CRISPR-Cas9 technology in gene editing lactobacillus is prospected.In the future,CRISPR-Cas9 technology will be combined with other gene editing methods to explore faster,more efficient and simple probiotic gene editing technology.

【Objective】 The aim of this study was to investigate the effects of RNA methyltransferase like 3 (METTL3) on the proliferation and differentiation of bovine skeletal muscle satellite cells (BSMSCs).【Method】 In this study,the growth and development of bovine skeletal muscle was simulated by the BSMSCs model induced differentiation in vitro.The difference of expression level of mRNA and proteins of METTL3 before and after differentiation of BSMSCs were detected by Real-time quantitative PCR (qRT-PCR) and Western blotting methods.The METTL3 interfering RNA (si-METTL3) was designed and synthesized,and the overexpression vector pcDNA3.1-METTL3 of METTL3 was constructed and transfected into BSMSCs respectively.The transfection effect and the expression of paired box 7 (Pax7) in BSMSCs were detected by qRT-PCR and Western blotting,and the cell proliferation was detected by EdU method.The BSMSCs transfected with si-METTL3 and pcDNA3.1-METTL3 were induced to differentiate in vitro,and the cell morphology of BSMSCs in the differentiation stage was observed by light microscope.The expression of myosin heavy chain (MyHC) and myogenin (MyoG),the differentiation marker of BSMSCs was detected by qRT-PCR and Western blotting.The m⁶A RNA methylation levels of BSMSCs at different periods after interference or overexpression of METTL3 were detected by colorimetry.【Result】 The results showed that after the differentiation of BSMSCs,the expression level of METTL3 protein was extremely significantly higher than that in the proliferation period (P＜0.01).After METTL3 was interfered or overexpressed,there was no significant difference in the mRNA and protein levels of Pax7 (P＞0.05),and neither of the EdU positive cell rate had no significant difference compared with the control group (P＞0.05).After interfering with the expression of METTL3,the number and diameter of cellular myotubes increased significantly,the levels of mRNA and protein expression of MyHC were extremely significantly higher than that of the control group (P＜0.01),the level of protein expression of MyoG was extremely significantly higher than that of the control group (P＜0.01),and the m⁶ A RNA methylation levels during BSMSCs proliferation and differentiation phase were extremely significantly downregulated(P＜0.01).After overexpressing of METTL3,the number and diameter of cellular myotubes were significantly reduced,the levels of mRNA and protein expression of MyHC were extremely significantly lower than that of the control group (P＜0.01),the level of protein expression of MyoG was extremely significantly lower than that of the control group (P＜0.01),and the m⁶ A RNA methylation levels during BSMSCs proliferation and differentiation phase were increased extremely significantly (P＜0.01).【Conclusion】 The METTL3 had no significant effect on the proliferation of the BSMSCs,but it could regulate the myogenic differentiation process of BSMSCs,and affect the m⁶ A mRNA methylation level of BSMSCs.Interfering with METTL3 could promote myogenic differentiation progression,and reduce the m⁶ A RNA methylation levels of BSMSCs. While overexpressing METTL3 could inhibit the myogenic differentiation progression,and increase the m⁶A RNA methylation levels of BSMSCs.The results provided the foundation for further exploring the regulatory mechanism of METTL3 in bovine muscle development.

【Objective】 Through sequencing and bioinformatics analysis of 4 polyembryony related genes in Inner Mongolia White cashmere goats,the single nucleotide polymorphisms (SNP) significantly related to litter size were deeply explored,so as to provide a theoretical basis for improving cashmere goat breeding efficiency.【Method】 244 cashmere goat ewes in type Alashan and Aerbasi were selected as candidates,and MultipSeq PCR combined with second-generation high-throughput sequencing technology was used to detect the polymorphism of growth differentiation factor 9 (GDF9),bone morphogenetic protein 15 (BMP15),bone morphogenetic protein receptor 1B (BMPR1B) and beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2) genes,the association between different SNPs and litter size of cashmere goats were analyzed.【Result】 A total of 172 SNPs were predicted by GATK analysis,including 95,33,26 and 18 SNPs related to BMPR1B,B4GALNT2,BMP15 and GDF9 genes,respectively.The correlation analysis results of GLM model showed that 10 SNPs were significantly correlated with the litter size (P＜0.05),and among them genotypes of 7 SNPs illustrated significantly related with litter size with further chi-square test (P＜0.05).The litter size of CC,GG,GA,CA and CC genotypes of BMPR1B gene at g.29893723 C＞G,g.29897064 G＞A,g.29897722 G＞A,g.29897734 C＞A and g.29938673 C＞G were significantly higher than that of CG,GA,GG,CC and GG genotypes (P＜0.05),respectively.The litter size of GG and GA genotypes of B4GALNT2 gene at g.37072289 G＞A were significantly higher than that of AA genotype (P＜0.05),and the litter size of CC and AC genotypes of GDF9 gene at g.66027842 A＞C were significantly higher than that of AA genotype (P＜0.05).【Conclusion】 The sequence characteristics of SNPs of GDF9, BMP5, BMPR1B and B4GALNT2 genes related to multiparity traits in Inner Mongolia White cashmere goats were obtained through the study,and a total of 10 SNPs were significantly related to litter size in cashmere goats,and revealed the difference among litter size of different genotypes.The results could provide a powerful reference SNP for molecular-assisted breeding of Inner Mongolia White cashmere goats.

【Objective】 This study was aimed to clone the coding region (CDS) sequence of neuronal pentraxin 1 (NPTX1) gene in Dolang sheep,analyze its structure and function by bioinformatics,and study its expression pattern in different tissues of gonadal axis during puberty initiation in Dolang sheep.【Method】 The hypothalamus,pituitary,oviduct,ovary and uterus tissues of healthy female Dolang sheep were collected from prepubertal,pubertal and postpubertal.The total RNA was extracted and reverse transcribed into cDNA.NPTX1 gene of Dolang sheep was amplified by PCR using prepubertal hypothalamus cDNA as template,cloned and sequenced,and its bioinformatics analysis was carried out.The expression of NPTX1 gene in hypothalamus,pituitary,ovary,uterus and oviduct of Dolang sheep were analyzed by Real-time quantitative PCR.【Result】 The cloned NPTX1 gene was 1 576 bp,of which the CDS sequence was 1 299 bp,and the similarity with the mRNA sequence of sheep predicted on GenBank(accession No.:XM_005683183.3) was 99.85%.Phylogenetic tree analysis results showed that NPTX1 gene of Dolang sheep had the closest genetic distance to Ovis aries and the farthest genetic distance to Gallus gallus.Bioinformatics analysis results showed that NPTX1 protein was an unstable hydrophilic acidic protein with a signal peptide and belonged to secretory protein.In the secondary structure of NPTX1 protein,alpha helix and random coil accounted for 41.90% and 40.73%,respectively,which were consistent with the predicted results of the tertiary structure.Real-time quantitative PCR results showed that NPTX1 gene was expressed in five different gonad tissues of Dolang sheep in three stages,and the expression was the highest in hypothalamus of puberty,which was significantly higher than that of prepuberty and postpuberty (P＜0.05).【Conclusion】 The experiment successfully cloned the full-length CDS sequence of NPTX1 gene in Dolang sheep,and revealed the differences in the expression of NPTX1 gene in gonadal axis tissue of Dolang sheep at different stages of puberty,which provided a basis for further exploring the regulation mechanism of NPTX1 gene in puberty onset of Dolang sheep.

The function and structure of brown adipocytes are quite different from white adipose adipocytes,brown adipocytes are rich in mitochondria.It is able to utilize excess energy metabolism substrates for thermogenesis.It is the main source of heat for non-shivering thermogenesis in young mammals,and it also plays an important role in maintaining the body’s energy balance.The thermogenesis process is mediated by uncoupling protein 1 (UCP1) on the inner membrane of cell mitochondria,and both the body’s environment and its response to hormones can affect the activity of brown adipose tissue.Upon stimulation,adrenergic receptors on the surface of brown adipocytes are activated,triggering an intracellular cascade that simultaneously enhances the expression of genes involved in thermogenesis,increases energy expenditure,and decreases fat deposition.Brown adipose tissue has become a potential therapeutic target for obesity and energy metabolism-related diseases.In view of the role of brown adipose tissue in fat deposition and energy metabolism,the research on brown adipose tissue in livestock and poultry is also more and more in-depth.The author summarized the relevant studies on ovine brown fat,mainly include environmental factors that affect the activity of ovine brown fat such as cold stimulation,nutrient restriction,chronic hypoxia and feeding additives,and hormonal factors (prolactin and its receptors,thyroid hormones,leptin,reproductive hormones),in order to provide a certain theoretical basis for the application of ovine thermogenic fat.

【Objective】 The purpose of this experiment was to screen safer and more efficient adjuvants for Porcine circovirus type 2 (PCV2) synthetic peptide vaccine.【Method】 The adjuvants of IMS 251C VG,GEL 02 PR,ISA 15A VG,ISA 206 VG,ISA 61 VG,Taiwan bacterial flagella,Carbomer,polysaccharide were used to make PCV2 synthetic peptide vaccine according to specific methods.The physical properties of each vaccine were verified,the safety and immune effect of the vaccine were preliminary tested by mouse test,and the better adjuvants were selected for immune challenge protection test of piglets to further evaluate the immune protection effect of each vaccine. 【Result】 The physical properties showed that all vaccines had good stability,and the storage period at 4 ℃ was more than 12 months.In terms of viscosity and emulsion droplet size,ISA 61 VG oil adjuvant was relatively higher and larger,and the two-phase adjuvant of ISA 15A VG and ISA 206 VG were second,the other water adjuvants were lower and smaller.The mouse test results showed that all adjuvants except Taiwan bacterial flagella adjuvant were safe.The results of mouse ELISA antibody level test showed that the immune effect of ISA 61 VG,GEL 02 PR and ISA 206 VG were better than other vaccines.Safety and immune effect were evaluated comprehensively,five adjuvant groups including ISA 61 VG,GEL 02 PR,Carbomer,polysaccharide and ISA 206 VG were selected for immune challenge protection test of piglets, and the results of immune challenge protection test of piglets showed that,the effects of ISA 61 VG and GEL 02 PR were obviously better than other adjuvants.The ELISA antibody and neutralizing antibody level produced by the vaccines prepared by them were significantly higher than those of other groups,and could also stimulate the production of higher levels of cytokine interferon-γ (IFN-γ).After challenge,the relative daily gain of piglets in ISA 61 VG,GEL 02 PR and ISA 206 VG groups was significantly higher than that in other adjuvant groups(P＜0.05),and the results of Real-time PCR and immunohistochemistry showed that the above vaccine groups could significantly reduce the viral load in piglets (P＜0.05).The observation of clinical symptoms showed that the two vaccine groups ISA 61 VG and GEL 02 PR had no temperature rise or roughened hair coats,and the pathological section showed that the pathological changes in the tissues of the two vaccine groups were mild.【Conclusion】 The results showed that the PCV2 synthetic peptide vaccine prepared by oil adjuvant ISA 61 VG and water adjuvant GEL 02 PR had better immune protection against PCV2 infection.This results provided a data basis for the study of PCV2 synthetic peptide vaccine.

【Objective】 This study was aimed to investigate the drug resistance of animal-derived Escherichia coli (E.coli) isolates in Hebei and the genetic relationship among the drug-resistant strains after the prohibition of resistance,so as to provide a scientific basis for the effective prevention and control of E.coli drug resistance.【Method】 297 fecal samples (48 from chickens and 249 from pigs) of healthy animals were collected from a farm in Hebei province.The isolates were identified by bacterial isolation and purification and PCR amplification,and the resistance of the isolates were determined by drug susceptibility test.Drug resistance gene detection,serotype identification,phylogenetic group analysis,plasmid replicon typing and genotyping were performed.【Result】 The isolates appeared as pink round colonies on MacConkey medium.A total of 129 strains of E.coli were isolated and identified by 16S rDNA sequencing analysis,of which 92 strains of E.coli showed multi-drug resistance,mainly 3-4 resistance,and the detection rate of the drug resistance genes were the highest with floR,tetA,qnrS and strB.The multidrug-resistant E.coli was dominated by O132 serotype,and the phylogenetic groups were mainly distributed in groups A and B1,and the main prevalent plasmids were IncFIB and IncN,27 ST types were obtained by MLST typing,mainly ST93 type,of which 12 sequence types were unknown type.【Conclusion】 The phenomenon of multi-drug resistance of E.coli in the intestine of healthy animals in Hebei was relatively serious,with numerous serotypes and diverse molecular characteristics,and it was still necessary to strengthen the monitoring of drug resistance and molecular characteristics of E.coli of animal origin.

【Objective】 This study was aimed to understand the molecular characteristics of Pseudorabies virus (PRV) strains and vaccine strains in Guizhou province,the genetic evolution of PRV variants in Guizhou province and the antigenic differences between PRV strains and vaccine strains were investigated.【Method】 gB,gC,gD genes of 5 PRV strains isolated from Guizhou province from 2015 to 2021 and commercial vaccine strains (C strains and HB2000 strains) were cloned and sequenced by molecular cloning technology and bioinformatics technology.Using the sequences of two PRV Guizhou strains in the sequence library of our laboratory and the vaccine strains registered on GenBank,systematic genetic evolution and differential analysis of B cell linear antigen epitopes were conducted.【Result】 The results showed that the genome-wide nucleotide similarities of gB,gC and gD genes of the 7 Guizhou strains and vaccine strains were 98.1% to 100%,94.7% to 100% and 98.8% to 100%,respectively.The genome-wide amino acid similarities of gB,gC and gD genes of the 7 Guizhou strains and vaccine strains were 96.4% to 100%,90.9% to 100% and 97.0% to 100%,respectively.Amino acid replacement analysis showed that there were 56,68 and 23 amino acid sites differences in gB,gC and gD genes between the 7 PRV Guizhou strains and vaccine strains,among which the amino acid sites of gC gene were the most different.GZQX2017 strain lacked gE gene.In the genetic evolution tree,GZQX2017 strain and Bartha-K61,Becker,NIA3 and other foreign strains were located in the same large evolutionary branch,belonging to PRV typeⅠ.The remaining six Guizhou strains inserted an aspartic acid (D) at the 48th and 496th positions respectively in gE gene,which was in line with the genetic characteristics of the epidemic variant,and located in another major evolutionary branch with the domestic epidemic PRV variant,belonging to PRV type Ⅱ.When the whole genome of GZQX2017 strain gB,gC and gD genes were used as target genes,Bartha-K61 strain was used as the main parent and Fa strain was used as the secondary parent,both gB and gC genes could detect the recombinant signal,but the recombinant signal of gD gene was not obvious.Compared with Bartha-K61 strain,there were differences in the number and sites of gB,gC and gD protein epitopes.【Conclusion】 Among the 7 PRV strains in Guizhou,GZQX2017 belonged to PRV type Ⅰ with vaccine Bartha-K61 and C strains. GZQX2017 might be the natural deletion of gE gene produced by recombination with Bartha-K61 as the main parent,which was expected to be used as a candidate vaccine strain for further study.The other 6 strains from Guizhou,Fa strain and HB2000 strain belonged to PRV type Ⅱ,which had the epidemic characteristics of epidemic mutant strains.This study was of great significance for understanding the molecular characteristics of PRV strains prevalent in Guizhou province,China.

【Objective】 The aim of this study was to clone the serine/threonine protein phosphatase 2A (Ser/Thr protein phosphatases 2A,PP2A) gene of Fasciola gigantica and construct a prokaryotic expression vector to obtain the prokaryotic expression protein,so as to lay a foundation for exploring the function of PP2A gene and its role in the development of Fasciola gigantica.【Method】 Total RNA was extracted from adult trematodes,the complete coding region of PP2A gene was amplified by RT-PCR and ligated to pET-28a(＋) vector by double restriction endonuclease digestion and sequencing,the positive plasmid was named as recombinant plasmid pET-28a-PP2A.The recombinant plasmid pET-28a-PP2A was transformed into E.coli expression strain BL21 (DE3) competent cell for IPTG induction,and the induction conditions of the recombinant protein were optimized.The expression effect of PP2A gene of Fasciola gigantica was detected by SDS-PAGE and Western blotting.Real-time PCR was used to detect the expression of PP2A gene in eggs,metacercaria,6-week-old juvenile and adults of Fasciola gigantica.【Result】 The coding region of PP2A gene of Fasciola gigantica was 1 161 bp,encoding 386 amino acids.The molecular formula was C₁₉₈₀H₃₁₀₅N₅₃₅O₅₆₆S₂₄ and the molecular weight was 44.23 ku.The results of bioinformatics analysis showed that the protein edited by PP2A gene was PP2Ac,the molecular function was signal transduction (GO:0004871),the biological process was protein dephosphorylation (GO:0006470),and the cell component was cytoplasm (GO:0005829).PP2A protein had no signal peptide and no transmembrane domain.It was a hydrophilic protein with 37 phosphate sites.The results of SDS-PAGE and Western blotting showed that the expression of PP2A in Fasciola gigantica was the highest at 30 ℃ and 0.8 mmol/L IPTG inducing for 6 h.The expressed product was mainly in the form of inclusion body,and the recombinant protein could be recognized by anti-His tag by Western blotting detection.The results of Real-time PCR showed that the expression of PP2A gene was the highest in the 6-week-old juvenile stage,which was extremely significant higher than that in other stages (P＜0.01),and the lowest in the metacercaria stage,and had no significant difference with the egg stage(P＞0.05).【Conclusion】 In this study,the prokaryotic expression vector of Fasciola gigantica PP2A was successfully constructed and the corresponding protein was obtained,which laid a foundation for studying the function of PP2A gene in the growth and development of Fasciola gigantica.

【Objective】 This study was aimed to obtain specific nanobody against signal transducer and activator of transcription 3 (STAT3) protein and then validate its use for immunohistochemistry and Western blotting,as well as the function on the cell growth of B16 melanoma cell.【Method】 Anti-STAT3 nanobodies were screened from B16 nanobody library from alpaca by phage display.After the positive clones were obtained and sequenced,one clone was ligated into the prokaryotic expression vector pColdⅠto construct the plasmid which was expressed by E.coli and purified by nickel columns,molecular sieves and ultrafiltration tubes.Immunohistochemistry and Western blotting were used to verify whether anti-STAT3 nanobody bound STAT3 protein in brain tissue and had the effects on the proliferation of B16 cells by CCK8 method and related genes expression on the proliferation pathway by Western blotting.【Result】 After phage display screening and sequencing of positive clones,a monoclonal strain of nanobody specifically binding STAT3 protein was successfully obtained and named as STAT3-VHH-10B.The plasmid of the ligation of STAT3-VHH-10B and pColdⅠwas expressed and then purified,which was verified by SDS-PAGE and showed anti-STAT3 nanobody with the size of about 15 ku.The results of immunohistochemistry and Western blotting showed that the purified anti-STAT3 nanobody could detect STAT3 expression in brain tissue.The results of cell proliferation test showed that anti-STAT3 nanobody could significantly inhibit the proliferation of B16 cells,and down regulate the protein expression of the main genes on the cell proliferation pathway,including CycD1, Bcl2 and cMyc.【Conclusion】 A specific anti-STAT3 nanobody was successfully screened and could be used for immunohistochemistry and Western blotting to detect the expression of STAT3 in tissues.Moreover,it could inhibit the proliferation of B16 cells by inhibiting the expression of main genes CycD1, Bcl2 and cMyc on the basis of binding with STAT3.The results would provide a new tool for the functional research of STAT3 and its nanobody.

Gut microbiota is one of the important parts of animal organism,and it has coexisted with the body in the long process of evolution.Many parasites also parasitize the intestinal tract of the host,gut microbiota and parasites interact with each other.Gut microbiota can not only affect the complex processes of parasites invading,colonizing and causing disease to the host,but also regulate the intestinal health of animals,and play an important role in antagonizing the infection of foreign pathogens such as bacteria,viruses and intestinal protozoa.It plays an important role in maintaining the homeostasis of intestinal environment.Gut microbiota can affect host immunity through itself and its metabolites,so as to regulate the body’s own inflammatory response and resist intestinal parasite infection.Gut microbiota can also affect parasite invasion through space occupying effect and changes in the structure of intestinal microflora.Existing studies have found that the effects of parasite infection on gut microbiota homeostasis can not only have negative effects such as mechanical injury and inflammatory reaction,but also have beneficial regulation on the host body.After infecting the host,some parasites can activate the body-related immune response and stimulate the body to release anti-inflammatory factors,which can help the intestinal tract resist bacterial infection and reduce the inflammatory response.The excretion and secretion of some parasites can affect the structure of gut microbiota in the host body and act as an immunomodulator.In recent years,the interaction mechanism between microbiota and host pathogen antagonism has become a hot spot,which has been widely concerned by scholars at home and abroad.As a result,parasite therapy and gut microbiota transplantation have also become a research hotspot.The metabolites of parasites and intestinal flora also have certain effects on the intestinal environment and homeostasis,but the specific mechanism of their effects is not clear.There are few reports on the interaction mechanism between parasites and gut microbiota in China.The author mainly reviewed the interaction between parasites and gut microbiota in order to provide reference for researchers to carry out relevant research.

【Objective】 This study was aimed to understand the antibiotic resistance,genome structure and degree of genetic variation of Riemerella anatipestifer,so as to provide a reference for studying of antibiotic resistance and genome of Riemerella anatipestifer.【Method】 PCR identification,antibiotic resistance detection and draft whole genome sequencing of a suspected strain of Riemerella anatipestifer were performed,this suspected strain was isolated from brain tissue of died ducks,and its genetic evolutionary analysis was performed based on 16S rDNA and housekeeping genes.【Result】 After quality control and filtering,the sequencing data was successfully assembled into a draft genome containing 38 contigs.Six different antibiotic resistance genes were detected using Abricate software and CARD online database,four of which were consistent with the antibiotic resistance phenotype.The gene sequence was submitted to MLST database,and a new ST type (ST93) was found.The results of 16S rDNA and housekeeping gene phylogenetic trees showed that the isolates were closely related to two Riemerella anatipestifer strains.【Conclusion】 A strain of Riemerella anatipestifer was isolated and identified,which was a multiantibiotic resistant strain and carries multiple antibiotic resistance genes in its genome.The gene variation of Riemerella anatipestifer was large,and there was a risk of cross regional epidemic transmission.In clinical medication,we should pay attention to rational medication,timely monitor the horizontal transfer of resistance genes,and conduct regular epidemiological investigation to prevent the cross regional epidemic of Riemerella anatipestifer.

【Objective】 The purpose of this experiment was to identify and master the epidemiological characteristics and biological characteristics of Bovine viral diarrhea virus (BVDV) in Liaoning province,and to provide theoretical basis and technical support for the prevention and treatment of BVDV.【Method】 The tissues suspected of BVDV infection from a cattle farm in Liaoning province were collected,Infectious bovine rhinotracheitis virus (IBRV),Bovine respiratory syncytial virus (BRSV),and Bovine parainfluenza virus type 3 (BPIV3) and BVDV gene-specific primers were identified by RT-PCR,and MDBK cells were inoculated with interstitial fluid to isolate the virus.After cytopathic observation of the cells,the virus was identified again by electron microscopy,indirect immunofluorescence assay (IFA),and the whole genome sequence of the virus was amplified by PCR and analyzed for genetic evolution.【Result】 The identification result of BVDV specific primer RT-PCR was positive.There was a band at 280 bp,which was consistent with the expected band size.The other pathogen specific primers did not amplify the band,which proved that other pathogens were not infected.The isolated strain did not show obvious cytopathic effect in MDBK cells.After virus purification,virus particles with a diameter of about 50 nm were observed by electron microscopy.The results of IFA showed that bright green fluorescence could be observed in the MDBK cells inoculated with the isolated virus strain,while no fluorescence was observed in the control cells.The isolated virus was named LN-1 strain.The size of the whole genome amplification sequence was 12 269 bp.Genetic evolution analysis showed that LN-1 strain was in the same branch as XC,SD-15 and ZM-95 strains,and the nucleotide similarity with XC strain was 96.2%,belonging to BVDV-1m type.【Conclusion】 In this study,one strain of 1m BVDV strain was successfully isolated,which was of great significance for the follow-up vaccine research and development,epidemiological investigation and pathogenesis research.

【Objective】 The purpose of this experiment was to express nonstructural protein 7 (NSP7) of Porcine reproductive and respiratory syndrome virus (PRRSV) in eukaryotic cells and analyze its bioinformatics,so as to speculate its function in the process of PRRSV replication.【Method】 The target gene was synthesized according to the sequence of NSP7 gene in GenBank database,and the P3×FLAG-CMV-NSP7 eukaryotic expression vector was constructed.The protein expression was verified by Western blotting after transient transfection of Marc-145 cells.The subcellular localization of NSP7 was performed by indirect immunofluorescence assay (IFA),and the physicochemical properties,structure and function of NSP7 were predicted by bioinformatics analysis software.【Result】 The eukaryotic expression vector P3×FLAG-CMV-NSP7 was successfully constructed.Western blotting results showed that the target band with a size of 2.7 ku was obtained,which confirmed that the recombinant vector was highly expressed in Marc-145 cells.IFA results confirmed that NSP7 protein was mostly distributed in the cytoplasm at the initial stage of PRRSV infection,and entered the nucleus from the cytoplasm with the extension of infection time.Bioinformatics analysis results showed NSP7 protein was encoded by 259 amino acids,the molecular formula was C₁₂₈₄H₂₀₂₀N₃₄₈O₃₇₉S₈,the molecular weight was 28 652.75 u,the theoretical isoelectric point was 5.88,and it was an unstable and hydrophilic protein.Structural prediction found that the secondary structure of NSP7 protein included alpha helix and beta turn accounted for 35.91% and 5.41%, respectively.Nuclear localization sequence analysis showed that NSP7 protein had a transmembrane sequence RLNKKKRRRMEAVGIF.【Conclusion】 The eukaryotic expression vector of PRRSV NSP7 was successfully constructed.NSP7 was distributed in cytoplasm at the initial stage of PRRSV infection and in nucleus at the later stage of PRRSV infection.It was found that NSP7 had nuclear localization sequence.

【Objective】 This study was aimed to screen some broad-spectrum Riemerella anatipestifer phages as candidates for therapeutic phage combinations.【Method】 The host range of 35 phages were determined by spot tests with different contents of phages.The methods of transmission electron microscope observation,thermal sensitivity and pH stability evaluation,determination of the best multiplicity of infection (MOI) in the plural,one-step growth curve drawing and sterilization experiment were used to analyze the morphological features and some biological characteristics of phage.【Result】 Phage vB_RanS_202 had the broadest cleavage spectrum among 35 phages,which could cleave 42% (42/100) of Riemerella anatipestifer strains including 6 serotypes of 1,2,6,10,11 and 13.The phage had a hexagonal head with approximately 60 nm in diameter and a long tail with 220 nm in length,which was not heat-resistant.The titer of phage decreased from 1.0×10⁷ PFU/mL to 1.8×10⁵-2.6×10⁵ PFU/mL after 80 min of 50 ℃ water bath or 20 min of 60 ℃.The phage could maintain its activity at pH 5.0 to pH 9.0.Using RAf488 as host bacteria,the optimal MOI of the phage was 0.01.One-step growth curve indicated that the latent period of phage was 60 min,the outbreak period was 75 min,and the outbreak quantity was 150 PFU/cell.The results of bactericidal experiment against RAf488 showed that phage had good germicidal efficacy when MOI was 0.004 to 0.016.【Conclusion】 Phage vB_RanS_202 had a wide lysis spectrum and the ability of bactericidal in liquid.The results of this study provided a reference for the subsequent screening of phage therapeutic strains.

【Objective】 The purpose of this study was to investigat the effect of Astragalus polysaccharide on inactivated vaccine against Aeromonas hydrophila on hybrid sturgeon,so as to explore the potential of Astragalus polysaccharide as a vaccine adjuvant.【Method】 A total of 140 healthy hybrid sturgeon were randomly divided into four groups (control group,vaccine group,Astragalus polysaccharide group and combined group) and subjected to corresponding immune treatment.Blood samples were collected on days 0,7,14,21 and 28,the antioxidant indexes (superoxide dismutase (SOD)) and immune related index (lysozyme (LYZ),acid phosphatase (ACP),immunoglobulin M (IgM) and complement C3(C3)) were detected.At the end of immunization,the virus challenge test was conducted to observe the death of sturgeon and the pathological changes of immune tissues.【Result】 On days 0,7,14,21 and 28,compared with control group,the activities of SOD,LYZ and ACP,and the contents of IgM and C3 in the serum of hybrid sturgeon in combined and vaccine groups were increased significantly or extremely significantly (P＜0.05 or P＜0.01).The serum SOD and LYZ activities,and IgM and C3 contents in Astragalus polysaccharide group were increased significantly or extremely significantly (P＜0.05 or P＜0.01),while the LYZ activity was no significant difference (P＞0.05).Compared with vaccine group,the activities of LYZ and ACP in serum of Astragulus polysaccharides group were significantly decreased (P＜0.05),the activity of SOD,and the contents of IgM and C3 were no significant difference(P>0.05).Compared with Astragalus polysaccharide group or vaccine groups,the serum SOD,LYZ and ACP activities in combined group were significantly increased (P＜0.05),the IgM content was significantly decreased (P＜0.05),and the C3 content was no significant difference (P ＞0.05).The combination of Astragalus polysaccharide and inactivated vaccine could effectively enhance the disease resistance of hybrid sturgeon,and the relative immune protection rate reached 100%. There was no obvious pathological damage to the liver,spleen and intestinal tissue of hybrid sturgeon.【Conclusion】 When Astragalus polysaccharide was combined with Aeromonas hydrophila inactivated vaccine for hybird sturgeon,it could effectively improve the antibody titer of the vaccine and enhance the protection after the inactivated vaccination,which inducing the immune response of hybird sturgeon and improving the disease resistance,and providing a theoretical reference for the prevention and control of fish diseases caused by Aeromonas hydophila.

【Objective】 The purpose of this study was to establish an ELISA method for detection of IgA antibody against Porcine epidemic diarrhea virus (PEDV).【Method】 In this study,PEDV epidemic strain CH/HNPJ/2017(MF152604.1)was used as the biological material,and the S2 gene (2 368—4 170 bp) was amplified by RT-PCR and inserted into prokaryotic expression vector pET-24a to transform the competent cells of Escherichia coli BL21 (DE3).It was induced by IPTG and purified by nickel column.Western blotting was used to verify the reactivity and specificity of recombinant protein S2 with PEDV positive serum.Using it as the coating antigen,an IgA antibody ELISA method based on recombinant full-length S2 protein of PEDV variant strain was established.The chessboard method was used to determine the optimal detection conditions,and its sensitivity,specificity and repeatability were determined.It was preliminarily applied to the detection of 130 porcine serum and 40 porcine oral mucus.【Result】 The S2 gene was amplified by RT-PCR,and the recombinant expression vector pET-24a-S2 was successfully constructed.The recombinant S2 protein was obtained after being transformed into Escherichia coli and expressed and purified by induction.SDS-PAGE results showed that the purified S2 protein had specific bands and no heterobands.Western blotting test showed that the protein had good reactivity with PEDV positive pig serum.The optimal coating conditions for recombinant S2 protein were 0.5 μg per well at 4 ℃ overnight,the optimal serum reaction conditions were 1∶160 dilution at 37 ℃ for 60 min,the best reaction conditions for HRP-conjugated antibody were 1∶5 000 dilution at 37 ℃ for 30 min,and the optimal color rendering time of TMB was 10 min under ambient temperatures.The sample was positive when the S/P value ≥ 0.04,it was negative when the S/P value ≤ 0.02,and it was suspicious when the S/P value was between 0.02 and 0.04.The ELISA method established in this study was used to detect the standard positive serum of swine fever virus,porcine circovirus,porcine reproductive and respiratory syndrome virus,pseudorabies virus and porcine deltacoronavirus.The S/P values were all less than 0.02,indicating that the method had good specificity.When the PEDV positive serum was diluted by 640 times,it was still positive by this method,indicating that this method had good sensitivity.This method was used for inter assay and intra assay,and the coefficient of variation were all less than 8% (within 10%),which shows that this method had good repeatability.130 pig sera were detected by this method,and the coincidence rate between the results and IDEXX IgA antibody detection kit was 90.77%.The detection results of 40 samples of pig oral mucus showed that the positive detection rate was 90.0% and the negative detection rate was 93.3%.【Conclusion】 In this study,the recombinant plasmid pET-24a-S2 was successfully constructed,and the recombinant S2 protein with high purity was obtained,which was used as coating protein to establish an ELISA method for IgA antibody based on full-length S2 protein.The method could effectively detect both serum and pig oral mucus type samples,and had strong sensitivity,high specificity,stability,and repeatability,which was of great clinical practical significance to the clinical diagnosis and prevention and control of PEDV.

【Objective】 This study was aimed to analyze and determine the pharmacodynamic material basis of anti-inflammatory effect of Phellodendron amurense and Acorus calamus mixture,and determine the possible target.【Method】 The anti-inflammatory effect of Phellodendron amurense and Acorus calamus mixture was tested by auricular swelling test in mice,and then the interaction (PPI) network diagram with inflammation-related proteins was analyzed by network pharmacology.Cytoscape 3.7.2 software was used to draw the network diagram of drug-active component-disease-common target,and GO function and KEGG pathway were enriched and analyzed.Autodock software was used to connect the main active ingredients with the core target. 【Result】 The inhibition rate of auricular swelling induced by xylene in mice by Phellodendron amurense and Acorus calamus mixture was 56.96%,which was significantly different from the model group (P＜0.05).Through network pharmacology analysis,a total of 30 active compounds of Phellodendron amurense and Acorus calamus mixture such as quercetin,berberine,and kaempferol were obtained,and 25 intersection targets of Phellodendron amurense and Acorus calamus mixture and inflammation were interleukin-6 (IL6),IL1β,and serine/threonine protein kinase (AKT1).GO enrichment analysis results showed that a total of 334 biological processes,13 cellular components,and 27 molecular functions were obtained.Biological processes involved positive regulation of nitric oxide biosynthesis process,positive regulation of apoptotic process,positive regulation of smooth muscle cell proliferation,and inflammatory response.Cellular components involved the extracellular space,extracellular region,and secretory granules.Molecular functions involved cytokine activity,enzyme binding,RNA polymeraseⅡtranscription factor activity,and ligand-activated sequence-specific DNA binding.KEGG results analysis yielded 82 pathway,and it was found that there might be some relationship between anti-inflammatory effects of Phellodendron amurense and Acorus calamus mixture and TNF signaling pathway,IL17 signaling pathway,lipids and atherosclerosis.【Conclusion】 Phellodendron amurense and Acorus calamus mixture had a good anti-inflammatory effect,and its anti-inflammatory mechanism might involve the regulation of gene and protein expression of different signaling pathways by multiple active components,indicating that Phellodendron amurense and Acorus calamus mixture exerted anti-inflammatory effects through multiple components,multiple pathways and multiple targets.

【Objective】 The aim of this study was to investigate the inhibitory effect of Acanthopanax senticosus aqueous extract (ASAE) on Mandarin ranavirus (MRV) and the immunomodulatory effects of Micropterus salmoides.【Method】 In this study,the inhibitory activity of ASAE on MRV in Siniperca chuatsi brain cell 3 lines (SCB3) was detected with different doses of ASAE (1.2,1.0,0.8,0.6,0.4 and 0.2 mg/mL) and MRV. 5‰ ASAE was added to the basal diet and fed Micropterus salmoides for 15 days,the phagocytosis effect of ASAE on Micropterus salmoides was analyzed by quantitative Real-time PCR and flow cytometry,and the protective effect of ASAE on MRV was determined by MRV infection test (1×10⁷ TCID₅₀/mL MRV,intraperitoneal injection).【Result】 The inhibition test results showed that MRV inhibition rates were 95.27%,91.36%,84.55%,60.05%,32.16% and 2.49% when the different concentrations of ASAE (1.2,1,0.8,0.6,0.4 and 0.2 mg/mL) were added into SCB3 cell lines.The quantitative Real-time PCR results showed that the ASAE induced significant up-regulation of cell phagocytosis pathway genes including interleukin-10 (IL-10),tyrosine-protein kinase (Lyn) and actin related protein 2/3 complex,subunit 5 (ARPC5), and Fcγ receptor Ⅰa (FcγR Ⅰa) in Micropterus salmoides (P＜0.05).The flow cytometry analysis showed that the ASAE significantly increased the phagocytosis ability of head kidney leukocytes of Micropterus salmoides (80.9% phagocytosis rate).The infection results showed that the relative protective rate of ASAE group was 30.48%.Compared with the control group,ASAE significantly delayed the process of infection and death of Micropterus salmoides. The virus copy numbers in brain,spleen,midgut and hindgut tissue of ASAE group were significantly decreased (P＜0.05).【Conclusion】 The results proved that ASAE significantly activated the phagocytic activity of Micropterus salmoides and played an excellent immunoregulatory role.Through the vitro and vivo tests,ASAE had excellent anti-MRV effect and could effectively prevent MRV infection.

【Objective】 This study was aimed to establish an indirect ELISA method using IPAJ protein encoded by virulence related genes of Salmonella Pullorum as antigen.【Method】 The IPAJ gene was amplified by PCR and connected to pCzn1 expression vector to transform into Escherichia coli BL21 (DE3) competent cells.The recombinant protein of IPAJ gene was induced to express by IPTG and purified,and the reactivity of the protein was verified by Western blotting.The purified IPAJ protein was used to immunize rabbits at 60 days of age for three times to prepare polyclonal antibodies.The antibody titer was detected by ELISA method.ELISA conditions of IPAJ protein as diagnostic antigen were optimized by square titration.An indirect ELISA method using IPAJ protein as diagnostic antigen was established and compared with the results of plate agglutination assay.【Result】 The IPAJ prokaryotic expression vector of Salmonella Pullorum was successfully constructed,and the recombinant protein was purified as inclusion body with good reactivity.Western blotting analysis results showed that IPAJ protein was successfully expressed at 32 ku,indicating good specificity.Indirect ELISA results showed that the titer of polyclonal antibody against IPAJ was 1∶25 600,indicating that polyclonal antibody was successfully prepared.The optimal condition for indirect ELISA using IPAJ protein as diagnostic antigen was preliminarily established as follows:The antigen coating concentration was 5 μg/mL,and 5% skim milk powder was used for 1.5 h,the optimum dilution of the primary antibody was 1∶250 (incubated for 1 h),the optimum dilution of the secondary antibody was 1∶10 000 (incubated for 1 h),and the color was dark for 15 min.Compared with plate agglutination assay results,the coincidence rate was 91.00%.【Conclusion】 The recombinant protein of Salmonella Pullorum IPAJ had good specificity and reactivity.The indirect ELISA method preliminarily established for the diagnosis of Salmonella Pullorum had certain practicability,which could be applied to clinical mass detection,and provide a basis for the establishment of a rapid diagnostic method.

【Objective】 The purpose of this experiment was to analyze the effective active ingredients of Radix Paeoniae Rubra on antioxidative stress and its molecular mechanism using network pharmacology and molecular docking technology.【Method】 Active ingredients and related targets of Radix Paeoniae Rubra were collected from TCMSP database and then converted into gene names using UniProt database.Targets related to oxidative stress were collected from GeneCards and OMIM databases,and the intersection targets of Radix Paeoniae Rubra and oxidative stress were obtained by Veeny online platform.Subsequently,protein-protein interaction (PPI) network map and a Radix Paeoniae Rubra-target-oxidative stress visualization network for topological analysis were established by STRING database and Cytoscape 3.8.0 software,and then key targets were identified.The active ingredients and antioxidant signaling pathways of Radix Paeoniae Rubra were determined by GO function and KEGG pathway enrichment analysis with Metascape database.AutoDock and PyMol softwares were used to perform molecular docking verification of the core target of Radix Paeoniae Rubra.【Result】 12 active ingredients in Radix Paeoniae Rubra were screened,including baicalein,beta-sitosterol,ellagic acid,stigmasterol,(+)-catechin,etc.There were 76 active ingredients targets in Radix Paeoniae Rubra and 4 873 oxidative stress targets,including 69 intersection targets,such as protein kinase B (AKT1),activator protein 1 (JUN),cellular tumor antigen p53 (TP53),tumor necrosis factor (TNF) and caspase-3 (CASP3), etc.The results of GO function and KEGG pathway enrichment analysis revealed that antioxidative stress of Radix Paeoniae Rubra might be related to the phosphatidylinositol-3-kinase-threonine-protein kinase (PI3K-Akt),neurofibromin-κB (NF-κB),and other signaling pathways.Moreover,the results of molecular docking showed that the main active ingredients of Radix Paeoniae Rubra,baicalein and beta sitosterol,had good binding ability to the core targets.【Conclusion】 The active ingredients such as baicalein,beta sitosterol and ellagic acid in Radix Paeoniae Rubra might play an antioxidative stress role by regulating key targets in PI3K-Akt,NF-κB,interleukin-17 (IL17) and other signaling pathways.

【Objective】 The aim of this experiment was to prepare monoclonal antibody to African swine fever virus (ASFV) p30 protein,and lay the foundation for the establishment of a rapid diagnostic test for ASFV and the research of a vaccine.【Method】 The p30 prokaryotic expression plasmid pET-30a(+)-p30 was constructed,transformed into E.coli BL21 (DE3) competent cells for induced expression,and then the target protein was obtained by nickel column affinity chromatography.p30 was compounded and immunized to BALB/c mice for three times every two weeks,and the spleen of immunized mice was fused with myeloma cells to prepare monoclonal antibody by ELISA method and subclonal screening of antibody-secreting hybridoma cells.The screened monoclonal cells were injected into the peritoneal cavity of mice to prepare ascites.The monoclonal antibodies were identified by Western blotting detection,immunofluorescence assay (IFA) and antibody isotype analysis.【Result】 The p30 protein prokaryotic expression plasmid was successfully constructed,and the recombinant p30 protein expressed in the prokaryotic system was purified using IPTG to induce.Four monoclonal antibody hybridoma cell lines,named 1H3B4,3F2F5,6E3A1 and 9D6B9,which could stably secrete the ASFV p30 protein,were successfully prepared.The potencies of the antibodies were determined by ELISA in the range of 1∶100 000 to 1∶1 000 000.The screened monoclonal antibodies were identified by Western blotting and IFA and those antibodies were specifically reacted with p30 protein.The monoclonal antibodies were identified as IgG2b for the heavy chain and Kappa for the light chain of 3F2F5 and 9D6B9.6E3A1 had IgM for the heavy chain and Kappa for the light chain.1H3B4 had IgG2b for the heavy chain and Lambda for the light chain.【Conclusion】 Four monoclonal antibodies against ASFV p30 protein were successfully prepared in this experiment,and the immunological characteristics of the monoclonal antibodies were analyzed,the results provided basis for the biological function research of p30 protein and the serological detection of ASFV.

【Objective】 The purpose of this study was to understand the distribution and prevalence and drug resistance of Riemerella anatipestifer (R.anatipestifer) in some areas of Hubei,and further to provide a reference for the clinical medication of this disease on duck farms in these regions.【Method】 The suspected cases of R.anatipestifer from meat duck farms in districts and counties around Wuhan (Jiangxia district,Huangpi district,Huangmei county) were sampled and the pathogenic bacteria were isolated.The morphology,dyeing characteristics,biochemical characteristics and 16S rRNA of the isolated bacteria were identified.The serotype and drug resistance of the isolated strains identified as R.anatipestifer were analyzed.The genomes of the isolated strains were identified by specific primer PCR of marker gene integrase to determine whether 50K gene island was integrated.【Result】 A total of three R.anatipestifer strains were identified.All the three strains could grow as the morphology of smooth translucent colonies on TSA plate medium.The results of Gram staining for the three isolated strains were negative,and the results of Wright staining showed bipolar dyeing.Biochemical test results showed that the three strains of R.anatipestifer did not ferment glucose,were catalase positive,did not produce hydrogen sulfide,and could not liquefy gelatin.16S rRNA identification results showed that the target band with a size of 1 480 bp was obtained,and the serotype was type 1.PCR amplification results showed that only the isolated HM strain contained the 50K genomic island.Minimum inhibitory concentration and Kirby-Bauer tests showed that these strains were sensitive to penicillin,amoxicillin,ceftriaxone,cefotaxime,florfenicol and gentamicin,and vary degrees of resistance to nalidixic acid,spectinomycin,enrofloxacin and polymyxin B.【Conclusion】 In this study,three strains of serotype 1 R.anatipestifer strains were isolated and identified,the results of drug sensitivity could provide some theoretical guidance for the prevention,treatment and epidemiological study of R.anatipestifer in these regions.

【Objective】 The aim of this study was to understand and master the pathogenicity of mixed infection of Micropterus salmoides with Aeromonas hydrophila (Ah),Aeromonas sobria (As) and Aeromonas veronii (Av),and screen out effective Chinese medicine for prevention and treatment of Aeromonas infection,so as to provide theoretical basis for prevention and treatment of mixed infection of Aeromonas.【Method】 Ah,As and Av were injected intraperitoneally into healthy Micropterus salmoides to establish the animal model of Micropterus salmoides infected by three Aeromonas alone and co-infected.The pathogenicity of Aeromonas co-infection was analyzed by organ index,histopathological section and mortality rate.The traditional Chinese medicines with inhibitory effect on Aeromonas in vitro was screened from 37 traditional Chinese medicines,and the key active ingredients and action targets of Fructus Mume-Scorpion double prescription in vivo were analyzed by network pharmacology.The therapeutic effect of Fructus Mume-Scorpion double prescription on Micropterus salmoides co-infected with three Aeromonas species was verified by medicinal bath method.【Result】 The pathogenicity of co-infection with three Aeromonas species on Micropterus salmoides was higher than that of infection alone.When infected alone,Ah caused the most severe organ damage,inflammation,and the highest mortality,followed by As and Av.Fructus Mume-Scorpion double prescription had significant inhibitory effect on Aeromonas in vitro,the antibacterial effect of the combination of Fructus Mume-Scorpion double prescription was stronger than that of single use.Through the analysis of network pharmacology,it was found that there were 180 common targets of action of Fructus Mume-Scorpion double prescription and bacterial infection,11 common targets such as tumor protein 53 (TP53),AKT serine/threonine-protein kinase 1 (AKT1) and so on which were potential key targets in the treatment of bacterial infection with Fructus Mume-Scorpion double prescription.The results of medicine bath experiment showed that Fructus Mume-Scorpion double prescription could significantly reduce the clinical symptoms,liver and intestinal inflammation and cell necrosis caused by three Aeromonas species co-infection,and the mortality rate of Micropterus salmoides co-infected with three Aeromonas species was decreased by 66.7%,which had a good therapeutic effect.【Conclusion】 The research indicated that the pathogenicity of Micropterus salmoides co-infection with three Aeromonas species was higher than that of single infection.Fructus Mume-Scorpion double prescription could significantly improve the survival rate of Micropterus salmoides co-infected with three Aeromonas species,the results provided a theoretical basis for the study of pathogenic mechanism and prevention and control measures of Aeromonas co-infection.

【Objective】 The pathogen of a case of endometritis of an adult female South China tiger (Panthera tigris amoyensis) in Hangzhou Safari Park was isolated and identified,and its biological characteristics were studied.【Method】 The uterine secretions of the tiger were collected aseptically and the pathogenic bacteria were identified by using bacterial isolation and purification,morphological observation,biochemical identification of Merier Virtek^® 2 COMPACT system,and 16S rDNA sequence analysis.The pathogenicity of the clinical isolate was detected by intraperitoneal inoculation model of mice,and the sensitivity of the isolates to 50 drugs was identified by the universal Kirby-Bauer (K-B) test.The selected sensitive drugs were combined with symptomatic treatment to continuously observe whether the disease recurred and the birth situation.【Result】 A Gram-negative short rod-shaped bacillus isolate was obtained from the uterine secretion of the tiger.The identification result of Merrier Vitek^® 2 COMPACT system’s Gram negative card showed that the clinical isolated strain was Morganella morganii,which could ferment glucose,decompose mannose,D-glucose and urease,phosphatase,ornithine decarboxylase γ-glutamyltransferase reaction was positive.The sequence comparison analysis showed that the 16S rDNA sequence had the highest similarity with Morganella morganii,more than 99%.The results further showed that the bacterium was Morganella morganii,and was named HNH2019.The drug sensitivity results showed that the bacterium was multi resistant to 33 drugs,including polymyxin B,amikacin,cotrimoxazole,medimycin,tetracycline and cefoperazone,moderately sensitive to 6 drugs,including cefotaxime,ceftriaxone and cefoxitin,and sensitive to 11 drugs,including tobramycin,lomefloxacin,enoxacin and florfenicol.The results of mouse pathogenicity analysis showed that the median lethal dose of the isolate inoculated intraperitoneally in ICR mice was 1×10^7.5 CFU.After treatment with sensitive drugs,the tiger recovered and did not recur for three consecutive years.By September 2022,the female tiger had been pregnant many times and had given birth to five cubs in total.【Conclusion】 In this study,a Morganella morganii isolate was proposed as a potential pathogen of the South China tiger.After treatment with sensitive drugs selected by drug sensitivity test,the tiger recovered from endometritis without recurrence.The results of the study provided case reference and data support for the prevention and control of similar diseases,suggesting that attention should be paid to the potential harm of Morganella morganii to animals.

【Objective】 This study was aimed to explore the drug-resistance elimination of Scutellaria baicalensis (S.baicalensis) on apramycin-resistant Escherichia coli (E.coli).【Method】 The alcohol extract of S.baicalensis was extracted,and the minimum inhibitory concentration (MIC) of alcohol extract of S.baicalensis against drug-resistant E.coli was measured by agar dilution method,and the elimination rate was obtained by drug-resistance elimination test and photocopying method.Then,the drug-resistant changes of E.coli before and after drug-resistance elimination were detected by broth dilution and disk diffusion,the changes of bacterial morphology were observed by scanning electron microscopy (SEM),and the changes of relative expression of drug-resistance related genes were detected by Real-time quantitative PCR.【Result】 MIC of alcohol extract of S.baicalensis against apramycin-resistant E.coli was 41.25 mg/mL.The elimination rate of alcohol extract of S.baicalensis against apramycin-resistant E.coli was 11.7%-17.0%.The MIC of E.coli were reduced from 256 μg/mL to 16-32 μg/mL after alcohol extract of S.baicalensis treatment.Scanning electron microscope observation found that the surface of E.coli in alcohol extract of S.baicalensis treatment group was damaged and the morphology was abnormal. Real-time quantitative PCR detection showed that the mRNA relative expression of genes ompF,fimA,phnE and fhuF in E.coli was extremely significantly increased by alcohol extract of S.baicalensis (P<0.01).【Conclusion】 Alcohol extract of S.baicalensis could eliminate the drug-resistance of apramycin-resistant E.coli by upregulating the expression of ompF,fimA,phnE and fhuF genes,damaging the surface and improving the susceptibility of E.coli.