粪肠球菌Ace重组蛋白的表达、纯化及黏附活性研究

发布时间：2018-04-23 11:41

  本文选题：粪肠球菌 + Ace　； 参考：《南华大学》2007年硕士论文

【摘要】： 目的:构建含粪肠球菌(Enterococcus faecalis,E.faecalis)ace基因的重组载体,在大肠杆菌M15中进行诱导表达,纯化表达产物并进行免疫活性分析,研究Ace蛋白在E.faecalis对胶原蛋白及HeLa细胞黏附过程中的作用,为探索Ace蛋白生物学功能及研发快速诊断试剂盒提供实验依据。 方法:用Premier Primer 5.0引物设计软件分析E.faecalis的ace基因序列,设计特异性引物,以E.faecalis标准株E.faecalis JH2-2为模板进行聚合酶链反应(PCR)扩增ace基因,将其亚克隆入原核表达载体pQE30中,构建重组质粒pQE30/Ace,然后转化至表达宿主菌M15中进行诱导表达,利用SDS-PAGE和Western blot进行分析和鉴定表达结果,Ni-NTA亲和层析柱纯化重组蛋白,BCA法测定纯化蛋白浓度。将纯化的Ace重组蛋白(rAce)免疫新西兰兔,用所得抗体对rAce的免疫活性进行分析。用荧光素FITC标记E.faecalis JH2-2进行黏附实验及黏附抑制实验,免疫荧光显微镜观察结果。 结果:PCR扩增得到大小约930bp的目的片断;构建的重组质粒经PCR、双酶切鉴定和测序鉴定证明其中插入片段为ace目的基因,测序结果与Genbank上登录序列完全一致;SDS-PAGE分析显示,在IPTG诱导下,重组工程菌表达了一相对分子质量(Mr)约为37kDa的目的蛋白条带,目的蛋白在菌体细胞内主要以可溶性蛋白形式存在;经Ni-NTA亲和纯化获得了较高纯度的重组蛋白;Western-blot检测其能与6×His单克隆抗体发生特异性反应。利用纯化的rAce免疫新西兰兔,间接ELISA法测定兔免疫血清特异性抗体效价在1:16 000以上;从E.faecalis细胞壁提取的Ace蛋白能与rAce免疫兔抗血清识别。黏附实验显示E.faecalis JH2-2能够黏附于胶原蛋白Ⅰ及HeLa细胞表面;黏附抑制试验显示rAce多克隆抗体具有抑制46℃培养的E.faecalis JH2-2对胶原蛋白Ⅰ及HeLa细胞的黏附作用,这种抑制作用随着血清稀释度的增加而减弱。 结论: 1.成功构建了pQE30/Ace原核表达载体,将其转化至E.coliM15后表达出了一相对分子量(Mr)约为37kDa的重组蛋白; 2. Ace重组蛋白具有较好的免疫原性,能刺激新西兰兔产生高效价的特异性抗体,并具有良好的免疫反应性,有望应用于E.faecalis快速诊断中; 3. Ace是一种具有黏附活性的蛋白,与E.faecalis致病性密切相关。
[Abstract]:Objective: to construct a recombinant vector containing Enterococcus faecalisus E. faecalisae gene and express it in Escherichia coli M15, purify the expression product and analyze its immunological activity, and study the role of Ace protein in the process of E.faecalis adhesion to collagen and HeLa cells. To explore the biological function of Ace protein and to develop a rapid diagnostic kit to provide experimental basis. Methods: Premier Primer 5.0 primer design software was used to analyze the ace gene sequence of E.faecalis, and specific primers were designed. The ace gene was amplified by polymerase chain reaction (PCR) using E.faecalis JH2-2 as template, and subcloned into the prokaryotic expression vector pQE30. The recombinant plasmid pQE30 / Acewas constructed, then transformed into the expression host strain M15 for induction expression. The expression results were analyzed and identified by SDS-PAGE and Western blot. The purified protein was purified by Ni-NTA affinity chromatography column. New Zealand rabbits were immunized with purified Ace recombinant protein rAce. the immunological activity of rAce was analyzed with the obtained antibodies. E.faecalis JH2-2 was labeled with fluorescein FITC for adhesion test and adhesion inhibition test. The results were observed by immunofluorescence microscope. Results the target fragment of 930bp was amplified by 1: PCR, and the recombinant plasmid was identified by PCR, double enzyme digestion and sequencing. The result of sequencing showed that the inserted fragment was ace target gene, and the result of sequencing was consistent with that of Genbank. Under the induction of IPTG, the recombinant engineering strain expressed a relative molecular weight (Mr) protein band of 37kDa, and the target protein existed mainly in the form of soluble protein in the cell. The recombinant protein with high purity was obtained by Ni-NTA affinity purification. Western-blot analysis showed that the recombinant protein could react specifically with 6 脳 His monoclonal antibody. The purified rAce was used to immunize New Zealand rabbits. The specific antibody titer of rabbit immunized by indirect ELISA was more than 1:16, and the Ace protein extracted from the cell wall of E.faecalis could be recognized with the antiserum of rAce. Adhesion assay showed that E.faecalis JH2-2 could adhere to the surface of collagen 鈪，

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