结核分枝杆菌rPstS1-HspX融合蛋白的表达、纯化及其免疫反应性分析

目的 构建结核分枝杆菌rPstS1-hspX (rph)融合基因及其原核表达载体pET-23b(+)-rPstS1-hspXpET-23b(+)-rph],表达、纯化rPstS1-HspX (rPH)融合蛋白,并分析其免疫反应性.方法 采用基因拼接技术将PstS1和HspX编码基因通过多肽接头(GSGSG)的DNA序列进行连接,构建融合基因rph.将融合基因定向克隆入原核表达载体pET-23b(+),构建重组原核表达质粒pET-23b(+)-rph.将重组质粒转化大肠杆菌E.coli BL21 (DE3) pLysE感受态细胞,IPTG诱导融合蛋白表达.SDS-PAGE和Western印迹法鉴定其表达情况.用镍离子鳌合亲和层析柱纯化融合蛋白,Western印迹法初步评价融合蛋白的免疫反应性.结果 融合基因rph及其原核表达载体pET-23b (+)-rph构建成功.融合蛋白rPH主要以可溶性非包涵体形式表达,相对分子质量为51 000,表达量约占菌体总蛋白的23％.经亲和层析后得到了纯度达92％的融合蛋白.Western印迹证实融合蛋白能与结核病阳性血清发生特异性免疫反应.结论 成功构建了原核表达载体pET-23b(+)-rph,获得了rPH融合蛋白,为rPH融合蛋白在结核病诊断中的应用提供了依据.

Expression, purification and immunoreactivity of recombinant Mycobacterium tuberculosis PstS1-HspX fusion protein

Objective To construct the Mycobacterium tuberculosis rPstS1-hspX (rph) fusion gene and its prokaryotic expression vector pET-23b (+)-rpstS1-hspX pET-23b (+)-rph],and to evaluate the immunoreactivity of the fusion protein PstS1-HspX rPH ] after expression and purification.Methods The fusion gene rph was constructed using gene splicing of DNA fragment that encodes a short polypeptide liner (GSGSG),then cloned into the prokaryotic vector pET-23b(+) to generate a recombinant plasmid,pET-23b(+)-rph.The recombinant plasmid was transformed into E.coli BL21 (DE3) pLysE cells.The expression of the histidine-tagged (His-Tag) fusion protein was induced with isopropyl-β-thiogalactopyranoside (IPTG),and verified with SDS- PAGE and Western blot.The fusion protein was purified by nickel affinity chromatography and evaluated for immunoreactivity with Western blot.Results The fusion gene rph and its prokaryotic expression vector pET-23b (+)-rph were successfully constructed.The expressed 51 000 fusion protein mainly existed in soluble form,accounting for about 23％ of total proteins in E.coli.After nickel affinity chromatography,the purity of rPH reached 92％.The immunoreactivity of rph to positive serum from tuberculosis patients was confirmed by Western blot.Conclusion The successful construction of prokaryotic expression vector pET-23b (+)-rph and expression of fusion protein rPH provides experimental evidence for use of rPH fusion protein in diagnosis of tuberculosis.