延边株气肿疽梭菌的分离鉴定及cctA基因的克隆和表达

发布时间：2018-02-10 11:51

本文关键词： 气肿疽梭菌分离鉴定 cctA基因原核表达　出处：《延边大学》2015年硕士论文　论文类型：学位论文

【摘要】：气肿疽是由气肿疽梭菌引起的一种急性、热性、败血性传染病,因其具有发病快、死亡率高的特点,给畜牧业的发展带来了巨大的经济损失。该病主要以皮下和深部肌肉处发生气性、炎性肿胀,按压有捻发音为典型特征。而继2006年之后吉林省延边州敦化市、延吉市依兰镇、三道镇相继出现牛气肿疽病例,这严重制约了延边州乃至全国畜牧业的发展。随着其易感群体的逐渐扩大,气肿疽梭菌也越来越多的威胁到了人类的安全。为了制备安全高效的新型气肿疽疫苗,控制本地区气肿疽病的发生,本研究对延边州延吉市采集的疑似气肿疽梭菌感染的牛肝脏、脾脏、肌肉等组织进行了分离,应用病原学检查、生化鉴定、动物试验及分子生物学鉴定的方法对该菌进行了鉴定。根据GenBank中已发表的气肿疽梭菌cctA基因,设计合成一对特异性引物,并以分离纯化的延边株气肿疽梭菌总DNA为模板扩增出cctA基因,并进行克隆载体的构建。利用生物信息学方法,对cctA蛋白的理化性质、结构功能域、蛋白质二级结构和三级结构及抗原表位等重要参数进行了预测和分析。将克隆质粒pMD 19-T-cctA进行双酶切后,与pEGX-4T-1原核表达载体进行连接,构建pGEX-4T-cctA原核表达重组质粒,并对其阳性克隆诱导表达,进行SDS-PAGE和Western blot分析。研究结果表明：本试验成功的对延边株气肿疽梭菌进行了分离,与参考序列(JQ692583)相比,共存在5处突变,核苷酸和氨基酸序列的同源性均为99.0%；该蛋白的理论等电点为6.79,不存在跨膜区和信号肽序列,二级结构以p-折叠和无规则卷曲为主,主要有5个抗原表位区域。经SDS-PAGE和Western blot分析显示,可见约44 kDa大小的片段,并具有良好的反应原性。此结果为延边地区气肿疽病的免疫和微生态防治提供了重要的参考依据,并为CctA蛋白功能的深入研究提供了参考,并为气肿疽梭菌单克隆抗体的制备及合成肽疫苗的研发奠定了一定的基础。
[Abstract]:Emphysematous gangrene is an acute, caused by Clostridium chauvoei heat, septic infectious disease, which is due to the onset of fast, high mortality, brought huge economic losses to animal husbandry. The disease mainly in the subcutaneous and deep muscles at the occurrence of gas, inflammatory swelling, press twist pronunciation is typical. And since 2006 in Jilin Prefecture of Yanbian Province, Dunhua City, Yanji City, Yilan Town, three towns have appeared in bovine emphysema cases, which seriously restrict the development of Yanbian and the national animal husbandry. With gradual enlargement of the susceptible population, carbonis shuttle bacteria also more and more threatening the safety of people. In order to model blackleg vaccine preparation is safe and effective, control the local blackleg disease, the study of suspected Clostridium chauvoei infection of bovine liver, collected in Yanji Yanbian City, spleen, muscle and other tissues were isolated, disease. The original examination, biochemical test, animal test methods and molecular biological identification of the identification of the bacterium Clostridium chauvoei. According to the cctA gene published in GenBank, designed a pair of specific primers, and the separation and purification of Yanbian strains of Clostridium chauvoei total DNA by cctA gene as template expansion, and to construct the cloning vector. Using bioinformatics methods, physical and chemical properties of the cctA protein, protein structure and function domain, two level structure and three stage structure and important parameters of antigen epitopes were predicted and analyzed. The cloned plasmid pMD 19-T-cctA was digested, and the prokaryotic expression vector pEGX-4T-1 to connect construction of pGEX-4T-cctA Recombinant Prokaryotic expression plasmid, and the expression of SDS-PAGE positive clones, and Western blot analysis. The results show that: the successful test of the Yanbian strain of Clostridium chauvoei were separated, and Compared with the reference sequence (JQ692583), there were 5 mutation, nucleotide and amino acid sequence homology was 99%; the protein isoelectric point is 6.79. There is no transmembrane domain and signal peptide sequence, the two stage structure with p- sheet and coil, there are 5 main antigenic epitope regions by SDS-PAGE and Western. Blot analysis showed that the visible about 44 kDa fragments, and has a good immunogenicity. The results provide an important reference for the immune blackleg disease in Yanbian area and micro ecological control, and for further research of CctA protein function for reference, and makes some the foundation for the development and preparation of synthetic peptide vaccine against Clostridium chauvoei monoclonal antibody.

【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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