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：《微生物学杂志》2008年03期

【摘要】：构建含粪肠球菌表面蛋白Ace保守序列A的重组载体,在大肠埃希菌M15中进行诱导表达、纯化表达产物,研究其黏附活性。以粪肠球菌标准株JH2-2为模板进行PCR扩增ace基因保守序列A,构建重组质粒pQE30/Ace,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,同时用纯化的Ace重组蛋白免疫新西兰兔,用所得相应的抗血清分别进行黏附和黏附抑制实验。结果可见,构建的重组质粒经酶切鉴定和测序鉴定证明其中插入片段为ace基因保守序列A,测序结果与Genbank上登录序列完全一致;SDS-PAGE分析显示,重组工程菌表达了一相对分子质量(Mr)约为37 ku的目的蛋白条带,Western blot检测其能与6×His单克隆抗体发生特异性反应。粪肠球菌JH2-2能够黏附于胶原蛋白Ⅰ表面;抗Ace多克隆抗体可抑制46℃培养的JH2-2对胶原蛋白Ⅰ的黏附,这种抑制作用与其稀释度呈负相关。构建的原核表达载体PQE30/Ace在E.coli M15中成功地表达,纯化的重组Ace蛋白具有胶原黏附活性。
[Abstract]:A recombinant vector containing conserved sequence A of Enterococcus faecalis surface protein (Ace) was constructed and expressed in Escherichia coli M15. The expression product was purified and its adhesion activity was studied. The conserved sequence of ace gene was amplified by PCR using the standard strain of Enterococcus faecalis JH2-2 as template. The recombinant plasmid pQE30 / Acewas constructed and transformed into the expression host strain M15 for induction expression. The recombinant protein was purified by Ni-NTA affinity chromatography column with SDS-PAGE and Western blot. New Zealand rabbits were immunized with the purified Ace recombinant protein. The adhesion and adhesion inhibition tests were carried out with the corresponding antiserum. The results showed that the constructed recombinant plasmid was identified by restriction endonuclease digestion and sequencing, and the inserted fragment was conserved by ace gene. The results of sequencing were consistent with those of Genbank and the results of SDS-PAGE analysis showed that the recombinant plasmid was a conserved sequence of ace gene. The recombinant engineering strain expressed a target protein band of about 37 ku, which could react with 6 脳 His monoclonal antibody by Western blot. Enterococcus faecalis JH2-2 could adhere to the surface of collagen 鈪，

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