gp10基因的原核表达及其联合异烟肼的体外抗结核菌活性

Prokaryotic expression of gp10 gene and antibacterial activity of gp10 gene combined with isoniazid to Mycobacteriumtuberculosisinvitro

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。

Objective To express the mycobacteriophage D29 lysin gp10in prokaryotic cells,and to investigate its antibacterial activities of alone and combined with isoniazid（INH）to Mycobacterium tuberculosis（MTB）in vitro.Methods The gp10 gene was amplified by PCR using the genome of mycobacteriophage D29 as a template and cloned into pET32a（＋）vector.The constructed vector pET32a（＋）-gp10 was identified by restriction enzyme digestion and nucleotide sequencing,and transformed to E.coli BL21（DE3）for expression.The minimal inhibitory concentration（MIC）of recombinant protein gp10 was detected by resazurin drugs combination microtiter assay（REDCA）for MTB standard strain H37 Rv and clinical isolate of multidrug-resistant MTB,the fractional inhibitory concentration index（FICI）was used to the antibacterial activity between INH and gp10 in MTB H37 Rv.Results The gp10 at a length of 1 479 bp was amplified by PCR,the recombinant plasmid pET32a（＋）-gp10 was digested by EcoRⅠ and NotⅠ for 5 900 and 1 479 bp bands,gp10 sequence analysis was consistent with the sequence in GenBank,and it expressed a target protein with a relative molecular mass about 75 200.The MICs of recombinant protein gp10 were 256mg·L^-1 against standard strain H37 Rv and clinical isolate of multidrug-resistant MTB.The FICI of combined INH and gp10 was 0.5for MTB H37 Rv.Conclusion The recombinant protein gp10 is expressed successfully,and it shows good antibacterial activity against MTB H37 Rv and clinical isolate of multidrugresistant MTB.Synergism in MTB H37 Rv is observed with gp10 combined with INHin vitro.