幽门螺杆菌ureA基因的克隆表达及其免疫原性的初步研究

发布时间：2018-07-10 19:58

  本文选题：幽门螺杆菌 + ureA　； 参考：《山东理工大学》2010年硕士论文

【摘要】： 幽门螺杆菌是导致人类多种消化道疾病的致病原,有广泛地传染性,临床难以进行彻底的治疗,利用免疫保护是预防和治疗幽门螺杆菌感染的重要方法。目的为研究幽门螺杆菌(Helicobacter pylori)尿素酶A亚单位的免疫原性和交叉免疫保护作用,本实验克隆了幽门螺杆菌26695菌株尿素酶A亚单位编码基因ure A;构建原核表达载体,表达并纯化了ureA蛋白；用纯化蛋白免疫家兔,制备抗血清,研究抗体特异性和交叉免疫保护作用。为进一步探讨ureA蛋白免疫保护作用及单克隆抗体用于治疗或预防幽门螺杆菌引发的相关疾病奠定基础。 方法(1)用PCR方法从幽门螺杆菌26695菌株基因组中扩增ureA基因片段；并将其克隆至pMD18-T Vector上,转化大肠杆菌DH5α,进行鉴定；再将目的基因引入pBV220-ILl表达载体中,构建pBV220-IL1-ureA表达重组载体,进行表达载体鉴定、基因序列测定和生物信息学比对。(2)温度诱导含重组载体pBV220-ILl-ureA的工程菌DH5α,表达目的蛋白；SDS-PAGE分析ureA蛋白表达结果；Western-blotting进行目的蛋白的鉴定；胶制备法大量分离纯化蛋白。(3)将纯化蛋白与弗氏佐剂混合免疫家兔,制备抗血清；ELISA法检测家兔抗血清效价及与免疫原蛋白UreA蛋白的结合特异性；利用竞争性结合实验鉴定兔抗血清与病人阳性血清交叉保护作用。 结果(1)PCR特异性扩增获得全长714bp目的片段,测序后与GenBank数据库比对,确定目的片段为幽门螺杆菌26695菌株ureA基因序列,同源性高达99%。 (2)获得pBV220-IL1-ureA原核重组表达质粒,工程菌经42℃热诱导,获得包涵体表达的ureA蛋白,融合蛋白分子量约为41kD,胶制备法获得了大量纯化蛋白,浓度约为1.Omg/mL。(3)将纯化的ureA蛋白用于家兔免疫,获得家兔抗血清,效价为1：1.28×104。ELISA法检测家兔抗血清与病人抗血清具有良好的竞争性。 结论(1)成功构建了pBV220-ILl-ureA重组质粒,插入序列经测序证实正确,无移码现象,与GenBank公布的基因序列高度同源。(2)成功表达并纯化了ureA蛋白,且其具有良好的免疫原性,为动物免疫奠定基础。(3)所获家兔抗血清与ureA蛋白具有良好的特异结合性。(4)竞争性实验显示,兔抗血清和人阳性血清具有交叉反应,能够产生竞争性的免疫保护作用。这为利用ureA多抗或单抗进行免疫预防和治疗幽门螺杆菌感染导致的疾病奠定了实验基础。
[Abstract]:Helicobacter pylori is the causative agent of many digestive tract diseases in human beings, which is widely contagious and difficult to be treated thoroughly. The use of immune protection is an important method to prevent and treat Helicobacter pylori infection. Objective to study the immunogenicity and cross-immune protection of urease A subunit of Helicobacter pylori (pylori), we cloned the urease A subunit encoding gene ure A from Helicobacter pylori 26695 strain and constructed a prokaryotic expression vector. The ureA protein was expressed and purified, the rabbit was immunized with the purified protein, and the antiserum was prepared to study the specificity of antibody and the protective effect of cross immunity. To further explore the immune protection of ureA protein and monoclonal antibodies used in the treatment or prevention of Helicobacter pylori related diseases. Methods (1) ureA gene fragment was amplified from the genome of Helicobacter pylori 26695 strain by PCR, cloned into pMD18-T vector, transformed into Escherichia coli DH5 伪 for identification, and then the target gene was introduced into pBV220-ILl expression vector to construct pBV220-IL1-ureA expression vector. Expression vector identification, gene sequencing and bioinformatics comparison were carried out. (2) temperature induced recombinant vector pBV220-ILl-ureA engineering strain DH5 伪, expressed target protein was analyzed by SDS-PAGE analysis ureA protein expression results were identified by Western-blotting. (3) immunized rabbits with purified protein and Freund's adjuvant. The antiserum was prepared to detect the titer of rabbit antiserum and its binding specificity to UreA protein. The cross protective effect of rabbit antiserum and patient positive serum was evaluated by competitive binding experiment. Results (1) the full-length 714bp fragment was amplified by PCR and compared with the GenBank database. The target fragment was confirmed to be the ureA gene sequence of Helicobacter pylori 26695 strain. (2) the prokaryotic expression plasmid pBV220-IL1-ureA was obtained. After heat induction at 42 鈩，

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