延边地区牛气肿疽流行病学调查及CctA基因真核表达载体的构建与表达

发布时间：2018-03-29 06:42

本文选题：延边地区　切入点：气肿疽梭菌　出处：《延边大学》2016年硕士论文

【摘要】：气肿疽(Clostridium chauvoei infection),病原为气肿疽梭菌,是一种急性、热性、败血性传染病。常见的典型症状为肌肉丰满部位发生炎性、气性肿胀,触压有捻发音,有一定的地区流行性、季节性,多发生在潮湿的山谷牧场、天气炎热的多雨季节。近几年吉林省延边州相继爆发牛气肿疽,给畜牧业和养殖业的发展带来了巨大的冲击和经济损失,同时也逐渐地威胁到人类的安全和生产。本研究根据NCBI上气肿疽梭菌细胞毒素A(CctA)基因序列,设计两对特异性引物。一方面,将采集延边地区165头份的牛血液样本,分别进行血涂片镜检和PCR检测,再应用统计学对各组数据分析。另一方面,采用疑似感染气肿疽梭菌的牛肝脏、脾脏、心脏及肌肉等组织进行分离,应用病原学检查、生化鉴定、动物实验、分子生物学方法进行鉴定。将PCR扩增的CctA基因克隆至pMD19-T simple载体,对测序正确的CctA基因序列分析,然后亚克隆至pVAX-1真核表达载体,将构建的pVAXl-CctA真核表达质粒转入Vero细胞中,通过间接免疫荧光(IFAT)和免疫印迹(Western blotting)检测CctA基因的表达产物。结果显示,PCR方法检测牛气肿疽的平均阳性率为15.2%,应用统计学分析表明,牛气肿疽感染率在不同县市、不同品种之间差异性不显著(P0.05)；而不同年龄、不同地理条件气肿疽感染率差异性显著(P0.05)；在个体农户的饲养方式中阳性率显著高于规模化的养殖方式(P0.01)；说明牛感染气肿疽梭菌主要受年龄段、地理条件、饲养方式等因素的影响较大。气肿疽梭菌扩增的CctA基因大小为501 bp,测序得到的CctA基因核苷酸与氨基酸序列与气肿疽梭菌ATCC10092菌株进行同源性比对分析,有3处点核苷酸突变,核苷酸序列同源性为99.4%；存在着2个位点的氨基酸特异性改变,氨基酸序列同源性为98.8%。转染的pVAX1-CctA组的细胞有绿色荧光,且pVAX1-CctA重组质粒表达产物大小约为19 kDa,表明pVAX1-CctA质粒在Vero田胞中成功表达了CctA蛋白。本研究更好地预测和分析了本地区分离株的信息,对预防和控制气肿疽提供了参考,为气肿疽的核酸疫苗研制奠定了深厚的基础。
[Abstract]:Clostridium chauvoei Infection.Clostridium chauvoei Infection.The pathogen is Clostridium chauvoei, which is an acute, hot and septic infectious disease. In recent years, cattle emphysema and gangrene have erupted in Yanbian Prefecture, Jilin Province, which has brought enormous impact and economic losses to the development of animal husbandry and aquaculture. In this study, two pairs of specific primers were designed according to the gene sequence of Clostridium emphysema cytotoxin (NCBI). On the one hand, 165 bovine blood samples were collected from Yanbian area. Blood smear examination and PCR examination were performed respectively, and then statistical analysis was used to analyze the data of each group. On the other hand, bovine liver, spleen, heart and muscle tissues suspected to be infected with Clostridium emphysema were isolated and pathologically examined. The CctA gene amplified by PCR was cloned into pMD19-T simple vector, the sequence of CctA gene was sequenced correctly, then subcloned into pVAX-1 eukaryotic expression vector. The constructed eukaryotic expression plasmid of pVAXl-CctA was transferred into Vero cells. The expression products of CctA gene were detected by indirect immunofluorescence (IFATT) and Western blotting. The results showed that the average positive rate of detection of bovine emphysema by PCR was 15.2g. The infection rate of emphysema in cattle was not significantly different among different species in different counties and cities, but in different ages. The difference of emphysema infection rate in different geographical conditions was significant (P 0.05), and the positive rate of emphysema infection rate in individual farmers was significantly higher than that in large-scale breeding method (P 0.01), which indicated that cattle infected with Clostridium emphysema were mainly affected by age and geographical conditions. The size of CctA gene amplified by C. emphysema was 501bp.The sequence of nucleotide and amino acid of CctA gene was compared with that of ATCC10092 strain of C. emphysema, and there were 3 point nucleotide mutations. The nucleotide sequence homology was 99.4, and there were two amino acid specific changes, and the amino acid sequence homology was 98.8. The transfected pVAX1-CctA cells had green fluorescence. The expression product size of pVAX1-CctA recombinant plasmid was about 19 kDa, which indicated that the CctA protein was successfully expressed by pVAX1-CctA plasmid in Vero field cells. The information of the isolated strains in the region was better predicted and analyzed in this study, which provided a reference for the prevention and control of emphysema. It lays a solid foundation for the development of nucleic acid vaccine for emphysema.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.23

【相似文献】