结核分枝杆菌RipB基因的克隆、表达及对藤黄微球菌生长的影响

Cloning,Expression and Biological Characterization of RipBfrom Mycobacterium tuberculosis

提取肺结核分枝杆菌基因组DNA,PCR扩增RipB基因并将其连接到表达载体pET-21a上,重组质粒转化大肠杆菌BL21(DE3)感受态细胞后,用IPTG诱导表达重组蛋白。SDSPAGE表明:重组RipB表达蛋白量约占菌体总蛋白量的40%,而其在37℃表达时主要为包涵体,在15℃表达时主要在可溶上清内;Ni柱一步亲和层析获得重组RipB;100pmol/L重组RipB可显著促进藤黄微球菌生长。

The gene of RipB was amplified by PCR from the genome of Mycobacterium tuberculosis （MTB） and was constructed into the expression plasmid pET-21a. After transformation the recombinant RipB expression vector into E. coli BL21 （DE3） competent cell, recombinant RipB was induced expression in E. coli by IPTG. SDS-PAGE analysis showed that recombinant RipB was about 40% of total bacterial protein, mainly expressed as inclusion bodies at 37 ℃, but mainly soluble at 15℃. Recombinant RipB was one-step purified by Ni-NTA affinity chromatography and obviously accelerated the proliferation of micrococcus luteus at 100 pmol/L.