分枝杆菌同源可控表达系统的建立及其应用

Development of mycobacterial inducible expression system and application for immunological diagnostics on tuberculosis

目的构建分枝杆菌可控表达载体，利用其过表达结核分枝杆菌（Mycobacterium tuberculosis，Mtb）的免疫保护性抗原，亲和层析纯化后分析免疫原性。方法应用PCR扩增耻垢分枝杆菌（Mycobacterium smegmatis，Ms）乙酰胺酶编码基因启动子（pACE），构建分枝杆菌可控表达载体pMF系列；克隆Mtb嵌合抗原编码基因并用乙酰胺进行诱导表达，以Ni^2＋亲和层析纯化重组蛋白；并用该重组嵌合抗原Ag856 A2对卡介苗（BCG）免疫的小鼠脾细胞进行体外刺激，以IFN-γ酶联免疫斑点技术（ELISPOT）分析其免疫原性。结果成功构建了分枝杆菌可控表达载体pMF系列，利用0．02％乙酰胺进行诱导可实现目标抗原在Ms中的高水平表达；同时利用引入的6×Ilis标签可方便实现重组抗原的一步纯化，且该同源表达重组抗原可诱导更高水平IFN-γ的分泌。结论以Ms乙酰胺酶编码基因启动子pACE为基础构建的分枝杆菌可控表达系统可实现Mtb抗原在Ms中的高水平表达与纯化，与大肠杆菌异源表达相比，该同源表达重组抗原具有更好的免疫原性。

Objective To develop mycobaeterial inducible expression vectors which permit to overexpress Mycobacterium tuberculosis （Mtb） immunodomlnant antigen, and to analyze its immunogenicity after purification by affinity chromatography. Methods The regulatory region of M. smegmatis（Ms） acetamidase（pACE） was obtained by PCR amplification, and was used as promoter to construct the mycobacterial inducible expression vectors, pMF series. The coding gene of Mtb chimeric antigen Ag856A2 which is a recombinant Ag85A with 2 copies of ESAT-6 inserted in its Ace I site and showed excellent immunogenicity in the animal experiments we described previously, was eloned into the pMF vector series, and was induced to express by the addition of acetamide. The recombinant protein expressed in the Ms was purified by the Ni2^＋ -NTA affinity chromatography, the resulted homologous recombinant antigen was added into the spleen cells separated from BCG vaccinated mice, and the immunogenicity was analyzed by the IFN-3, ELISPOT assay. Results The mycobacterial inducible expression vectors, pMF series was constructed successfully, target antigen could be induced to express in the Ms by the addition of 0.02% acetamide, and could be purified by the Ni2^＋ -NTA affinity chromatography due to the addition of 6 × His tag in the vector pMF406. Furthennore, the mycobacterial homologous antigen could induce more IFN-γ secretion than the heterogonous one. Conclusion The mycobacterial inducible expression system based on the regulatory region of Ms acetamidase as promoter could permit the Mtb target antigen of interest overexpression and purification, and the immunogenicity of the homologous antigen from Ms is better than that of be expressed from E. coli, which may be more potential for immunological detection of tuberculosis.