幽门螺杆菌UreI免疫特性及其治疗性DNA疫苗的初步研究

发布时间：2018-05-08 14:31

  本文选题：幽门螺杆菌 + 尿素膜通道蛋白(UreI)　； 参考：《四川大学》2006年博士论文

【摘要】：幽门螺杆菌(Helicobacterpylri，Hp)是引起人急、慢胃炎、消化道溃疡、胃癌和反流性食道炎的病原体，其与人冠心病、过敏性紫癜等疾病密切相关。Hp能在人胃酸性环境定植，主要是因该细菌有尿素酶和尿素膜通道蛋白(UreI)。Weeks等2001年研究指出Hp的ureI的基因是Hp在胃酸性环境定植和生存的关键基因，推测其为抗Hp感染的新药物靶点，但目前没有实验证明。本研究用分子克隆方法获得Hp的ureI基因和其原核表达载体，再诱导得到重组UreI蛋白，并研究其免疫原性和免疫反应性。在ureI的5’端引入霍乱毒素B亚单位(CtB)基因，获得CtB与UreI融合的重组蛋白和真核表达载体。用Hp感染的小鼠模型评价该真核载体作为治疗性DNA疫苗的效果，探讨Hp的ureI作为新药靶点的可能性，，同时寻找有希望的治疗性疫苗。为达到此目的，本研究分五部分完成： 第一部分 Hp尿素膜通道蛋白(UreI)的基因克隆、原核表达和免疫特性分析 首先克隆得到幽门螺杆菌尿素膜通道的全长基因ureI，构建原核重组质粒pET32a(+)-ureI和其原核表达工程菌株，并在特定的条件下用IPTG诱导工程菌表达了分子量分为43kD带His标签的重组蛋白rUreI，经免疫印迹证明该重组蛋白的免疫反应性。 第二部分 ctB与ureI双基因融合的原核表达、蛋白纯化和免疫特性分析 克隆了霍乱弧菌B亚单位基因(Cholera Toxin B，ctB)，并引入ureI基因的5’端，构建原核重组质粒pET32a(+)-ctB／ureI和其原核表达工程菌株，并在特定的条件下经IPTG诱导工程菌表达了分子量为58kD带
[Abstract]:Helicobacter pylori (Helicobacter pylori) is the causative agent of acute, chronic gastritis, gastrointestinal ulcer, gastric cancer and reflux esophagitis. It is closely related to human coronary heart disease, allergic purpura and other diseases. The main reason is that the bacteria has urease and urea-membrane channel protein UreI. Weeks et al. In 2001, it was pointed out that the ureI gene of HP is the key gene of HP colonization and survival in gastric acid environment. It is supposed to be a new drug target against HP infection, but it has not been proved by experiments. In this study, the ureI gene of HP and its prokaryotic expression vector were obtained by molecular cloning, then the recombinant UreI protein was induced, and its immunogenicity and immunoreactivity were studied. Cholera toxin B subunit (CTB) gene was introduced into the 5'terminal of ureI to obtain the recombinant protein and eukaryotic expression vector of CtB and UreI fusion. To evaluate the efficacy of the eukaryotic vector as a therapeutic DNA vaccine, to explore the possibility of HP ureI as a new drug target, and to search for a promising therapeutic vaccine with a mouse model of HP infection. In order to achieve this goal, this study is divided into five parts: Part I: gene cloning, prokaryotic expression and immunological characterization of HP urea membrane channel protein UreI First, the full-length ureI gene of Helicobacter pylori urea membrane channel was cloned, and the prokaryotic recombinant plasmid pET32a( pET-ureI) and its prokaryotic expression engineering strain were constructed. The recombinant protein rUreI with 43kD labeled with His was induced by IPTG under certain conditions. The immunoreactivity of the recombinant protein was proved by Western blot. Part two: prokaryotic expression, protein purification and immunological characterization of ctB / ureI fusion The B subunit gene of Vibrio cholerae, Cholera Toxin, was cloned, and the 5 'end of ureI gene was introduced to construct the prokaryotic recombinant plasmid pET32a (pET-ctBrureI) and its prokaryotic expression engineering strain. The engineering strain was induced by IPTG to express the 58kD band under certain conditions.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

【参考文献】

相关期刊论文 前2条

1 王正祥;幽门螺旋菌的分子生物学研究现状[J];微生物学通报;1996年04期

2 吴利先,杨致邦,林珊珊,刘淼;幽门螺杆菌HpaA-CtxB融合蛋白的表达及其免疫原性研究[J];中国免疫学杂志;2004年05期

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