鼠伤寒沙门氏菌菌毛FimA与PefA蛋白的原核表达及纯化

发布时间：2018-08-19 13:39

【摘要】：沙门氏菌病又名副伤寒。有些沙门氏菌血清型可以引起牛沙门氏菌病。目前由于抗生素的长期滥用导致沙门氏菌耐药性普遍存在,不仅影响该病的防治,更对公共卫生造成威胁。随着规模化、集约化舍饲养牛方式的不断发展,牛沙门氏菌病成为阻碍其健康发展和影响食品安全的一个因素。沙门氏菌Ⅰ型菌毛无论是在菌毛装配上还是在基因、氨基酸序列方面,都较为保守。fimA是编码Ⅰ型菌毛主要的菌毛亚单位。菌毛Pef具有良好的特异性。pefA为其主要的菌毛亚单位。本试验通过传统检测方法对宁夏地区牛源沙门氏菌进行分离鉴定并确定了沙门氏菌的优势血清型;然后探索四对特异性引物用于PCR快速检测的可行性;最后利用原核表达的方法得到纯化的鼠伤寒沙门氏菌上述两种菌毛主要亚单位蛋白,以期望在后续的抗原性分析、诊断以及预防鼠伤寒沙门氏菌病提供参考依据。基于上述研究内容作了以下三个方面的工作:1宁夏地区牛源沙门氏菌的分离鉴定为了分析宁夏地区引起奶牛临床乳房炎、隐性乳房炎、犊牛腹泻、成母牛流产的病原体。本试验通过采集病料,利用一系列微生物诊断方法和分子生物学方法对样品中沙门氏菌进行分离鉴定。结果表明四种特异性引物PCR扩增结果与预期一致,可以用于后续沙门氏菌PCR快速检测的建立。627份病牛样品中有33份鉴定为沙门氏菌,分离率为5.26%。乳样中检出7种沙门氏菌血清型,组织样中检出2种沙门氏菌血清型,均为我国常见血清型。其中鼠伤寒沙门氏菌为优势血清型,其次为纽波特沙门氏菌。2鼠伤寒沙门氏菌菌毛FimA、PefA蛋白的原核表达为了获得鼠伤寒沙门氏菌菌毛FimA、PefA的重组蛋白。本阶段试验首先对两种菌毛亚单位进行B细胞表位分析,设计并合成引物,通过PCR技术扩增目的基因。利用T4连接酶构建重组质粒。将构建好的重组质粒导入到大肠杆菌中,并对诱导条件(IPTG浓度、诱导时间)进行优化。最后利用SDS-PAGE分析重组蛋白的表达形式。结果表明两种重组载体全部构建成功,重组蛋白全部表达。两种重组蛋白FimA、PefA相对分子质量分别约为36kDa、33kDa且与预期一致,说明表达的蛋白是目的蛋白。重组蛋白FimA和PefA在大肠杆菌内部积累,FimA蛋白主要以包涵体形式存在,而PefA蛋白主要以可溶形式存在。3菌毛亚单位蛋白FimA、PefA的纯化为了进行后续的抗原性分析需要纯化的重组蛋白FimA、PefA。利用Ni-AgaroseResin分离纯化FimA、PefA重组蛋白。利用Western blot对纯化的FimA、PefA重组蛋白进行分析。蛋白电泳和Western blot都显示为一条清晰的蛋白条带。结果显示它们的大小分别约为36 kDa、33 kDa。本试验获得纯度较高的重组蛋白FimA、PefA可以为进一步抗原性分析以及研发鼠伤寒沙门氏菌的疫苗和检测提供原材料。
[Abstract]:Salmonellosis is also known as paratyphoid fever. Some salmonella serotypes can cause bovine salmonellosis. At present, drug resistance of Salmonella is widespread due to the long-term abuse of antibiotics, which not only affects the prevention and treatment of the disease, but also poses a threat to public health. With the development of scale and intensive feeding of cattle, bovine salmonellosis has become a factor that hinders its healthy development and affects food safety. Salmonella type I fima is the main fima subunit encoding type I pili, both in its assembly and in gene and amino acid sequence. Pef has a good specificity. PefA is the main subunit of its pili. In this experiment, the traditional detection method was used to isolate and identify Salmonella from cattle in Ningxia area, and the predominant serotype of Salmonella was determined, and the feasibility of using four pairs of specific primers for rapid detection of Salmonella was explored. Finally, the purified protein of the two main subunits of Salmonella typhimurium mentioned above were obtained by prokaryotic expression, in order to provide reference for the subsequent antigenicity analysis, diagnosis and prevention of Salmonella typhimurium. In order to analyze the pathogens of clinical mastitis, recessive mastitis, diarrhea of calves and abortion of adult cows in Ningxia, the following three aspects of work were done: 1) isolation and identification of Bovine Salmonella from Ningxia area in order to analyze the pathogens of clinical mastitis, recessive mastitis, diarrhea of calves and abortion of adult cows. A series of methods of microbiological diagnosis and molecular biology were used to isolate and identify Salmonella in the samples. The results showed that the results of PCR amplification with four kinds of specific primers were consistent with expectations, and could be used for the rapid detection of salmonella by PCR. 33 out of 627 samples of infected cattle were identified as Salmonella, and the isolation rate was 5.26. Seven Salmonella serotypes were detected in milk samples and two Salmonella serotypes were detected in tissue samples, all of which were common serotypes in China. Among them, Salmonella typhimurium was the dominant serotype, followed by Newport Salmonella. 2. The prokaryotic expression of FimA PefA protein of Salmonella typhimurium in order to obtain the recombinant protein of FimA PefA from Salmonella typhimurium pili. In this stage, B cell epitopes of two subunits were analyzed, primers were designed and synthesized, and the target genes were amplified by PCR. The recombinant plasmid was constructed by using T4 ligase. The constructed recombinant plasmid was introduced into E. coli and the induction conditions (IPTG concentration, induction time) were optimized. Finally, the expression of recombinant protein was analyzed by SDS-PAGE. The results showed that the two recombinant vectors were constructed successfully and the recombinant proteins were all expressed. The relative molecular weight of the two recombinant proteins FimA PefA was about 36 kDa and 33kDa respectively, which indicated that the expressed protein was the target protein. The recombinant protein FimA and PefA accumulated in E. coli mainly in the form of inclusion body, while the PefA protein mainly existed in the form of soluble form. The recombinant protein was purified by Ni-AgaroseResin. Western blot was used to analyze the purified FimAminopefA recombinant protein. Both protein electrophoresis and Western blot showed a clear protein band. The results showed that their sizes were about 36 kDa and 33 kDa. respectively. In this study, the recombinant protein FimA PefA with high purity could be used as a raw material for further antigenicity analysis and the development of vaccine and detection of Salmonella typhimurium.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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