牛病毒性腹泻/黏膜病(1型+2型)、牛传染性鼻气管炎、牛副流感(3型)三联灭活疫苗(E2蛋白+C1株+HB01株)的研制与免疫效果评价

采用CHO细胞表达牛病毒性腹泻/黏膜病病毒1型（BVDV1）E2蛋白,采用杆状病毒重组表达牛病毒性腹泻/黏膜病病毒2型（BVDV2）E2蛋白,采用MDBK细胞微载体悬浮培养技术培养牛传染性鼻气管炎病毒（IBRV）和牛副流感病毒3型（BPIV3）,收获蛋白表达产物和细胞培养物,经纯化、灭活后与605佐剂混合,制备牛病毒性腹泻/黏膜病(1型+2型)、牛传染性鼻气管炎、牛副流感(3型)三联灭活疫苗(E2蛋白+C1株+HB01株)。将疫苗免疫健康易感牛进行免疫效果评价,结果表明该产品免疫效果良好,免疫牛IBRV和BPIV3中和抗体效价均可达到1∶77以上;BVDV1和BVDV2 E2蛋白琼扩抗体效价均可达到1∶32以上;免疫牛攻毒保护率均可达到4/5以上。     

4 株致病性大肠杆菌致犊牛腹泻的分离鉴定及毒力与耐药性分析

[目的]为查明致北疆地区某牧场犊牛腹泻的病原,对脱水濒死10日龄新生犊牛进行剖检,无菌采集充血肠系膜淋巴结2 份。[方法]通过采用培养、染色镜检、生化特性考察、PCR鉴定等方法来分离鉴定致病菌,并对分离致病菌株进行相关毒力基因检测及药物敏感性试验。[结果]经分离鉴定分离出4 株大肠杆菌,毒力基因PCR检测结果K99、Sta、irp2、Eae基因分别在163 bp、314 bp、301 bp、552 bp处有清晰条带,与预期[目的]片段大小相符;药物敏感性试验结果表明,分离株对头孢哌酮、链霉素、庆大霉素敏感,对恩诺沙星、卡那霉素高度敏感。[结论]犊牛腹泻要以预防为主,治疗建议采用对因治疗,并配合提高抵抗力的中兽药进行。     

猪流行性腹泻病毒S2基因B细胞表位的筛选及其单克隆抗体的制备与鉴定

目的 应用生物学软件与单克隆抗体技术相结合的方法鉴定PEDV S2基因B细胞表位肽。方法 利用CLC Sequence viewer 6.8软件及在线数据库IEDB筛选出PEDV S2基因B细胞表位,并人工合成表位肽。将表位肽与钥孔血蓝蛋白（KLH）耦联后作为抗原,免疫雌性BALB/c小鼠,通过ELISA法筛选出抗体效价较高的小鼠进行1次加强免疫,3 d后取脾脏制备脾细胞悬液进行细胞融合。经HAT选择培养基培养筛选有效杂交瘤细胞,采用ELISA法筛选阳性克隆继续进行扩大培养。将部分阳性杂交瘤细胞进行小鼠腹腔注射,并收集腹水。通过ELISA方法分别检测小鼠腹水和单克隆细胞株培养上清液抗体效价,确定最高效价作为后备细胞株。结果 筛选出B细胞表位肽序列为:MQYVYTPTYYML;免疫多肽抗原后融合前血清抗体效价达到1:2 000;BALB/c小鼠腹水及单克隆细胞株培养物上清液ELISA检测结果显示抗体效价达到1:4 000。结论 筛选出了PEDV S2基因B细胞表位,为PEDV表位肽疫苗载体构建研究提供参考。     

牛病毒性腹泻病毒E0-E2基因融合腺病毒的构建及小鼠免疫效果评价

【目的】 研究牛病毒性腹泻病毒（Bovine viral diarrhea virus,BVDV）E0和E2串联基因重组腺病毒作为基因工程疫苗的应用潜力。【方法】 采用PCR扩增、E0-E2基因融合并构建重组穿梭质粒pDC316-E0-E2,将其与AdMax腺病毒系统的骨架质粒共转染HEK293T细胞包装成重组腺病毒,通过Western blotting进行验证,并通过Reed-Muench法测定病毒滴度,通过肌内、皮下免疫接种小鼠后用ELISA方法及流式细胞检测进行免疫效果试验。【结果】 成功扩增到E0、E2基因目的片段,大小分别为681和1 122 bp,得到了完整的腺病毒Ad5-E0-E2;测定其滴度为1.1×10¹⁰ PFU/mL;Western blotting检测结果显示,Ad5-E0-E2外源基因在HEK293T细胞中表达,得到了与预期相符的目的条带（65 ku）;ELISA检测结果表明,通过肌内和皮下注射Ad5-E0-E2均能产生较高的抗体水平;流式细胞检测显示首免、二免后肌内和皮下注射Ad5-E0-E2组CD4^＋、CD4^＋/CD8^＋比值均极显著高于PBS对照组（P<0.01）。【结论】 本试验成功构建重组腺病毒Ad5-E0-E2,且具有较好的反应原性和免疫原性,能诱导机体产生针对BVDV的特异性抗体。     

几个植物种叶片生化物质对杉木种子的发芽效应

对杉木、丝栗栲及木荷叶片的生化物质进行了初步的分离,并用杉木种子进行了生物检测.结果表明:经木荷和丝栗栲处理的,就绝对发芽率和胚根长而言,其弱极性生化物质组分的促进作用大于极性生化物质组分的;就鲜质量和干质量而言,极性生化物质组分的促进作用大于弱极性生化物质组分的;经杉木处理的,就绝对发芽率、胚根长及鲜质量而言,杉木叶片弱极性生化物质组分的抑制作用大于极性生化物质组分的活性,而对于干质量而言,极性生化物质组分的抑制作用大于弱极性生化物质组分的.     

无抗发酵饲料对生长育肥猪生长性能、肠道菌群和养分表观消化率的影响

本试验旨在研究无抗发酵饲料对生长育肥猪生长性能、肠道菌群和养分表观消化率的影响。选择18 kg左右断奶仔猪60头,随机分为2个组,每组3个重复,每个重复10头猪。对照组饲喂含抗生素基础饲粮(每千克含黄霉素5 mg),试验组饲喂80%基础饲粮和20%的无抗发酵饲料组成的试验饲粮。试验期154 d。结果表明:1)试验组的末重、平均日增重和平均日采食量都显著高于对照组(P0.05),而料重比则显著低于对照组(P0.05)。2)试验组中期和后期的乳酸菌数量的对数值显著高于对照组(P0.05),而试验组中期和后期的大肠杆菌和沙门氏菌数量的对数值显著低于对照组(P0.05)。3)在十二指肠肠段,试验组肠道食糜中的乳酸菌和大肠杆菌数量的对数值显著高于对照组(P0.05)。4)在十二指肠肠段,试验组肠道每克和每平方厘米黏膜的乳酸菌数量的对数值显著高于对照组(P0.05);试验组肠道每克黏膜的大肠杆菌数量的对数值显著高于对照组(P0.05)。在空肠肠段,试验组肠道每克和每平方厘米黏膜的乳酸菌数量的对数值显著高于对照组(P0.05)。5)试验组的粗蛋白质和粗纤维的表观消化率显著高于对照组(P0.05)。由此可见,无抗发酵饲料能提高生长育肥猪的生长性能,改善肠道微生物平衡,增强消化能力。     

Genetic characterization of bovine viral diarrhea virus strains in Beijing,China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5''-untranslated region (5''-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-α (IFN-α), IFN-β, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-α, and IFN-β mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.     

Identification of novel tumour‐associated antigens in canine mammary gland tumour

Canine mammary gland tumour (MGT) is the most common neoplasm in female dogs and has similar biological characteristics to human MGT. Spontaneous canine MGT is a more attractive clinical model in oncological research than that of the murine experimental model. Tumour‐associated antigens (TAAs), which are produced in tumour cells, are applied as tumour markers, tumour vaccine antigens and molecular targets of therapeutic drugs. In this study, we have primarily identified 13 different TAAs of canine MGT by serological immunoscreening of cDNA expression library. The results of serological mini‐arrays of identified antigens showed that CCDC41 antigen specially reacted with 35% of sera from MGT‐dogs and did not react with control sera. We also found that HSPH1 mRNA expression levels increased significantly in MGT tissues. These findings will contribute to the development of diagnostic technologies and translational target therapies for dogs. Clinical relevance : HSPH1, which is strongly expressed in the tumour tissue, will be a possible vaccine antigen of canine MGT.     

Isolation,Identification and Analysis of Serotype and Antibiotic Susceptibility of Pathogenic Salmonella from Chickens

To investigate the situations of predominant strain and antibiotic resistance of pathogenic Salmonella from chickens in Guangxi Zhuang Autonomous region, Salmonella was pre-enriched and isolated from tissues of clinical suspected chickens, and the Salmonella isolates were identified by biochemical test using ID 32E System for the identification of Enterobacteriaceae of VITEK System ATB Expression, serotypes were determined by slid agglutination test, antibiotic susceptibility was analyzed by ATB VET Susceptibility Test Strip of Enterobacteriaceae antibiotics.The results showed that 34 Salmonella strains were obtained from 310 clinical samples, of which 1 strain belonged to A serogroup, 1 to C2 serogroup, 15 to B serogroup, 14 to D serogroup and 3 to untyped serogroup, and S.typhimurium of B serogroup and S.pullorum of D serogroup were the predominant serotypes.All Salmonella isolates were sensitive to meropenem, imipenem, spectinomycine and apramycine, and the resistance rates to chloramphenicol, kanamycine and gentamicine were less than 10%, and the resistance rates to amoxicillin, streptomycine, flumequine, oxolinique, sulfamethizol, tetracycline and nitrofurantoine ranged between 50% and 90%, while the resistance rates to penicilline, oxacilline, fusidique, rifampicine and metronidazole reached to 100%.The results indicated that the serotype distribution of pathogenic Salmonella from chickens exhibited regional characteristics, and S.typhimurium and S.pullorum were the predominant serotypes in Guangxi Zhuang Autonomous region, and antibiotic resistance of Salmonella isolates was very serious which should be highly concerned.