粪肠球菌Ace重组蛋白的表达、纯化及黏附活性研究  

Expression and Purification of Enterococcus faecalis Ace Recombinant Protein and Its Adherence Activity

作　　者：武楠[1] 颜向军[2] 刘亚琴[2] 邹礼衡[2] 杨祚升[1] 

机构地区：[1]南华大学附属第一医院检验科 [2]南华大学附属第一医院血液科,湖南衡阳421001

出　　处：《微生物学杂志》2008年第3期34-39,共6页Journal of Microbiology

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。In order to construct a recombinant vector containing a gene encoding Ace A domain of Enterococcua faecalis （EF）, and induced express in E. coli M15 then purify its expression product to study its adherence activity, the Ace A domain was amplified by PCR from the genome of EF JH2-2, subcloned into the expression vector pQE30 to generate recombinant plasmid pQE30/Ace, then transferred and expressed in E. coli M15, and analyzed and identified the expression results by SDS-PAGE and Western blot. The recombinant protein was purified by Ni-NTA affinity chromatography, and immunized New Zealand rabbits with the purified Ace recombinant protein, and carry out adherence and adherence inhibition assay with obtained antiserum respectively. The results of restriction enzyme digestion analysis and sequencing showed that the inserted target gene was Ace domain identical with what reported in GenBank; SDS-PAGE analysis showed that the recombinant engineered strain expressed a relative molecular weight （Mr） at 37 ku target protein band, Western blot tested it could specifically react with 6 × His monoclonal antibody. EF JH2-2 was able to adhere to the surface of type Ⅰ collagen. Antibody to Ace A domain were showed to inhibit the binding of EF JH2-2 cultured at 46 ℃ to type Ⅰ collagen, and their dilutions had negative correlation with the inhibition. Therefore, prokaryotic expression vector pQE30/Ace was constructed and expressed successfully in E. coli M15. And the purified recombinant Ace protein has the adherence activity to collagen.

关 键 词：粪肠球菌 ACE 重组蛋白 蛋白纯化 黏附 

分 类 号：Q939.93[生物学—微生物学]