【摘要】：目的:原核表達分枝桿菌噬菌體D29內溶素gp10蛋白,檢測其單用及與異煙肼聯合對體外結核菌的抗菌活性。方法:采用PCR法從分枝桿菌噬菌體D29基因組中擴增gp10基因,克隆至原核表達載體pET32a(+),經雙酶切和測序鑒定正確的重組質粒轉化至大腸桿菌BL21(DE3)中表達目的蛋白。采用微量刃天青顯色法測定gp10蛋白對結核分枝桿菌(MTB)標準株H37Rv和臨床分離耐多藥株的最低抑菌濃度(MIC),同時觀察其與異煙肼聯用對H37Rv的部分抑菌濃度指數(FICI)。結果:PCR擴增獲得長1 479bp的gp10基因,重組質粒pET32a(+)-gp10經EcoRⅠ和NotⅠ雙酶切獲得長約5 900和1 479bp的條帶,gp10基因測序結果與GenBank中基因序列一致,并表達相對分子質量約75 200的目的蛋白。重組蛋白gp10對MTB H37Rv和臨床分離耐多藥株的MIC為256mg·L-1,其與異煙肼聯用對H37Rv的FICI為0.5。結論:成功表達重組蛋白gp10,gp10對體外MTB H37Rv和臨床耐多藥株均表現出良好抗菌活性,其與異煙肼聯用對體外H37Rv表現出協同效應。
[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，

本文編號：2421919