副溶血性弧菌FlaE蛋白的原核表达、纯化及其多克隆抗体制备

发布时间：2018-03-12 13:09

本文选题：副溶血性弧菌　切入点：flaE基因　出处：《暨南大学》2011年硕士论文　论文类型：学位论文

【摘要】：副溶血性弧菌(Vibrio parahaemolyticus, VP)是存在于近岸海洋环境的嗜盐细菌,它常常因为污染海味食品而引起人类急性胃肠炎,也可引起反应性关节炎,是一种重要的食源性致病菌。副溶血性弧菌具有极性鞭毛和周身鞭毛两个鞭毛系统以适应不同环境,其鞭毛蛋白有良好的免疫原性,可作为抗原制备抗体。本实验通过构建原核表达极性鞭毛FlaE蛋白的重组大肠杆菌,经IPTG诱导表达并通过亲和层析纯化获得目的蛋白,以其作为抗原制备多克隆抗体,为制备免疫磁珠,建立VP的快速富集和检测奠定基础。本实验主要得研究内容和结果如下：经Genebank中查得Vibrio parahaemolyticus,BB22菌株极性鞭毛基因FlaE基因序列(登录号为U12817.2),设计引物,利用PCR方法采用高保真酶从标准株ATCC17802基因组中扩增出flaE基因片段,回收所得的扩增产物与pMD 19-Tvector连接,转化大肠杆菌DH5a,利用氨苄青霉素、蓝白斑筛选和菌落PCR初步确认阳性重组子,随后进行测序鉴定。测序结果表明目的基因全长1122bp,与Genebank上VP. BB22 flaE基因片段同源性达98%。双酶切pMD 19-T重组子,再与经相同双酶切处理的表达载体pET22b(+)连接,得到重组表达载体pET22b-flaE,转化大肠杆菌BL21(DE3)。0.8 mM IPTG,37℃,3h诱导重组蛋白表达。经SDS-PAGE分析表明蛋白分子量约为40kDa(与DNASTAR计算结果一致),以包涵体形式表达。对目的蛋白的多聚组氨酸标签进行亲和层析纯化目的蛋白,SDS-PAGE电泳分析纯化结果,同时利用BCA法测定融合蛋白含量。纯化蛋白经佐剂乳化制备抗原,免疫新西兰兔子,收获抗血清,用ELISA方法测得抗体效价为1：204800.
[Abstract]:Vibrio parahaemolyticus (VPP) is a halophilic bacterium found in the coastal marine environment. It often causes acute gastroenteritis and reactive arthritis in humans by contaminating seafood. Vibrio parahaemolyticus has two flagellum systems, polar flagellum and peri-flagella to adapt to different environments, and its flagellin has good immunogenicity. The recombinant Escherichia coli expressing polar flagellum FlaE protein was constructed and expressed by IPTG. The target protein was purified by affinity chromatography and used as antigen to prepare polyclonal antibody. In order to prepare immunomagnetic beads and establish the rapid enrichment and detection of VP, the main contents and results of this experiment are as follows:. The FlaE gene sequence of polarity flagella gene of Vibrio parahaemolyticus bb22 strain (accession number U12817.2) was identified by Genebank. Primers were designed to amplify flaE gene fragment from the standard ATCC17802 genome by PCR method. The recovered amplified product was linked to pMD 19-T vector. E. coli DH 5a was transformed into E. coli, positive recombinant was identified by ampicillin, blue and white spot screening and colony PCR. The result of sequencing showed that the target gene had a total length of 1122 BP, and the homology with the VP. BB22 flaE gene fragment on Genebank was 98%. The recombinant pMD 19-T was digested by double enzyme, and then ligated with the expression vector pET22b (), which was treated with the same double enzyme digestion. The recombinant expression vector pET22b-flae was obtained and transformed into E. coli BL21(DE3).0.8 mM IPT GG for 3 h to induce the expression of recombinant protein. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was about 40 kDa (consistent with the result of DNASTAR calculation and expressed as inclusion body). The polymorphic of the target protein was expressed in the form of inclusion body. The purification result of the target protein was analyzed by SDS-PAGE electrophoresis. At the same time, the fusion protein was determined by BCA method. The purified protein was emulsified with adjuvant to prepare antigens, immunize New Zealand rabbits and harvest antiserum. The titer of the purified protein was 1: 204800 by ELISA method.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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