gp10基因的原核表达及其联合异烟肼的体外抗结核菌活性

发布时间：2019-02-13 21:59

【摘要】：目的:原核表达分枝杆菌噬菌体D29内溶素gp10蛋白,检测其单用及与异烟肼联合对体外结核菌的抗菌活性。方法:采用PCR法从分枝杆菌噬菌体D29基因组中扩增gp10基因,克隆至原核表达载体pET32a(+),经双酶切和测序鉴定正确的重组质粒转化至大肠杆菌BL21(DE3)中表达目的蛋白。采用微量刃天青显色法测定gp10蛋白对结核分枝杆菌(MTB)标准株H37Rv和临床分离耐多药株的最低抑菌浓度(MIC),同时观察其与异烟肼联用对H37Rv的部分抑菌浓度指数(FICI)。结果:PCR扩增获得长1 479bp的gp10基因,重组质粒pET32a(+)-gp10经EcoRⅠ和NotⅠ双酶切获得长约5 900和1 479bp的条带,gp10基因测序结果与GenBank中基因序列一致,并表达相对分子质量约75 200的目的蛋白。重组蛋白gp10对MTB H37Rv和临床分离耐多药株的MIC为256mg·L-1,其与异烟肼联用对H37Rv的FICI为0.5。结论:成功表达重组蛋白gp10,gp10对体外MTB H37Rv和临床耐多药株均表现出良好抗菌活性,其与异烟肼联用对体外H37Rv表现出协同效应。
[Abstract]:Aim: to express the gp10 protein of Mycobacterium bacteriophage D29 in prokaryotic expression and to detect its antibacterial activity against tuberculous bacilli in vitro and in combination with isoniazid. Methods: the gp10 gene was amplified by PCR from the genome of Mycobacterium phage D29 and cloned into prokaryotic expression vector pET32a (),. The recombinant plasmid was transformed into E. coli BL21 (DE3) by double enzyme digestion and sequencing. Determination of minimal inhibitory concentration of gp10 protein against Mycobacterium tuberculosis (MTB) standard strain H37Rv and clinical isolates of multidrug resistant strains by microchromogenic method (MIC), simultaneously observed the partial inhibitory concentration index (FICI).) of H37Rv combined with isoniazid Results: the length of 1 479bp gp10 gene was amplified by PCR. The recombinant plasmid pET32a ()-gp10 was digested with EcoR 鈪，

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