本文選題：幽門螺桿菌 + 尿素膜通道蛋白(UreI)�。� 參考：《四川大學(xué)》2006年博士論文

【摘要】：幽門螺桿菌(Helicobacterpylri，Hp)是引起人急、慢胃炎、消化道潰瘍、胃癌和反流性食道炎的病原體，其與人冠心病、過敏性紫癜等疾病密切相關(guān)。Hp能在人胃酸性環(huán)境定植，主要是因該細(xì)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在胃酸性環(huán)境定植和生存的關(guān)鍵基因，推測其為抗Hp感染的新藥物靶點(diǎn)，但目前沒有實(shí)驗(yàn)證明。本研究用分子克隆方法獲得Hp的ureI基因和其原核表達(dá)載體，再誘導(dǎo)得到重組UreI蛋白，并研究其免疫原性和免疫反應(yīng)性。在ureI的5’端引入霍亂毒素B亞單位(CtB)基因，獲得CtB與UreI融合的重組蛋白和真核表達(dá)載體。用Hp感染的小鼠模型評價該真核載體作為治療性DNA疫苗的效果，探討Hp的ureI作為新藥靶點(diǎn)的可能性，，同時尋找有希望的治療性疫苗。為達(dá)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、原核表達(dá)和免疫特性分析 首先克隆得到幽門螺桿菌尿素膜通道的全長基因ureI，構(gòu)建原核重組質(zhì)粒pET32a(+)-ureI和其原核表達(dá)工程菌株，并在特定的條件下用IPTG誘導(dǎo)工程菌表達(dá)了分子量分為43kD帶His標(biāo)簽的重組蛋白rUreI，經(jīng)免疫印跡證明該重組蛋白的免疫反應(yīng)性。 第二部分 ctB與ureI雙基因融合的原核表達(dá)、蛋白純化和免疫特性分析 克隆了霍亂弧菌B亞單位基因(Cholera Toxin B，ctB)，并引入ureI基因的5’端，構(gòu)建原核重組質(zhì)粒pET32a(+)-ctB／ureI和其原核表達(dá)工程菌株，并在特定的條件下經(jīng)IPTG誘導(dǎo)工程菌表達(dá)了分子量為58kD帶
[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.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R392

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相關(guān)期刊論文 前2條

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