脑膜炎奈瑟氏菌表面蛋白NspA基因的克隆与原核表达

发布时间：2018-06-17 23:55

本文选题：脑膜炎奈瑟氏菌 + NspA基因　；参考：《长春理工大学》2011年硕士论文

【摘要】：脑膜炎奈瑟氏菌是流行性脑膜炎的病原菌,根据其荚膜多糖抗原不同可分为13个血清群,包括A, B, C, D, X, Y, Z,29E, W125, H, I, K,根据细菌外膜蛋白和多脂糖成分不同可再分为血清型与亚型。我国流行菌群以A群为主,其次为B群和C群。近年来B群致病率有上升的趋势。1997年Matin等报道,脑膜炎球菌的外膜上存在一种低分子量的保守的表面蛋白命名为脑膜炎奈瑟菌表面蛋白NspA (Neisseria surface Protein), NspA能在菌体持续表达,抗NspA单克隆抗体能保护小鼠免于致死量脑膜炎奈瑟氏菌的攻击,用重组的NspA免疫小鼠能诱导免疫保护,是最具有潜力的脑膜炎奈瑟氏菌疫苗的候选抗原,渴望用于制备成防治流脑的疫苗。本文以NspA基因为目的基因,参照Genbank中已有的序列和原核表达质粒PMD18-T, pSIP-409的序列,按照其开放读码框架,设计引物,体外扩增目的基因,并将目的基因插入到PMD18-T, pSIP-409,得到重组质粒PMD18-T-NspA, pSIP-409-NspA.并对PMD18-T-NspA基因的序列进行测定,进一步将目的基因与pSIP-409-NspA载体连接转入大肠杆菌表达目的基因,实现NspA重组蛋白在大肠杆菌中的高效表达。
[Abstract]:Neisseria meningitidis is the pathogen of epidemic meningitis. According to its capsule polysaccharide antigen, it can be divided into 13 serum groups. It includes A, B, C, D, X, Y, ZN29E, W125, H, I, K, and can be subdivided into serotype and subtype according to bacterial outer membrane protein and fatty sugar composition. Group A was the most prevalent group in China, followed by group B and group C. In recent years, the pathogenicity of group B has been increasing. In 1997, Matin et al reported that there was a low molecular weight conserved surface protein on the outer membrane of Neisseria meningitidis called Neisseria surface protein. Anti-NspA monoclonal antibody can protect mice from the lethal dose of Neisseria meningitidis. Immunizing mice with recombinant NspA can induce immune protection and is the most potential candidate antigen of Neisseria meningitidis vaccine. Desire to be used to prepare a vaccine for the prevention and treatment of meningitis. According to the sequence of prokaryotic expression plasmids PMD18-T, pSIP-409 in Genbank, primers were designed to amplify the target gene in vitro and inserted into PMD18-T, pSIP-409. the recombinant plasmids PMD18-T-NspAand pSIP-409-NspAwere obtained. The sequence of PMD18-T-NspA gene was sequenced. The target gene was further ligated with pSIP-409-NspA vector to express the target gene in E. coli, and the recombinant protein of NspA was highly expressed in Escherichia coli.
【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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