CAJ | 학술논문

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 tu-berculosis,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Ag856A2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×His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,与大肠杆萧异源表达相比,该同源表达重组抗原具有更好的免疫原性.
목적 구건분지간균가공표체재체,이용기과표체결핵분지간균(Mycobacterium tu-berculosis,Mtb)적면역보호성항원,친화층석순화후분석면역원성.방법 응용PCR확증치구분지간균(Mycobacterium smegmatis,Ms)을선알매편마기인계동자(pACE),구건분지간균가공표체재체pMF계렬;극륭Mtb감합항원편마기인병용을선알진행유도표체,이Ni~(2+)친화층석순화중조단백;병용해중조감합항원Ag856A2대잡개묘(BCG)면역적소서비세포진행체외자격,이IFN-γ매련면역반점기술(ELISPOT)분석기면역원성.결과 성공구건료분지간균가공표체재체pMF계렬,이용0.02%을선알진행유도가실현목표항원재Ms중적고수평표체;동시이용인입적6×His표첨가방편실현중조항원적일보순화,차해동원표체중조항원가유도경고수평IFN-γ적분비.결론 이Ms을선알매편마기인계동자pACE위기출구건적분지간균가공표체계통가실현Mtb항원재Ms중적고수평표체여순화,여대장간소이원표체상비,해동원표체중조항원구유경호적면역원성.
Objective To develop mycobacterial inducible expression vectors which permit to overexpress Mycobacterium tuberculosis (Mtb) immunodominant antigen, and to analyze its immunogenicity after purification by affinity chromatography. Methods The regulatory region of M. smegmatis (Ms) acet-amidase(pACE) was obtained by PCR amplification, and was used as promoter to construct the mycobacteri-al 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 Acc Ⅰ site and showed excellent immunogenicity in the animal experiments we described previously, was cloned 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 Ni~(2+)-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-γ ELISPOT as-say. 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 puri-fied by the Ni~(2+) -NTA affinity chromatography due to the addition of 6×His tag in the vector pMF406. Fur-thermore, the mycobactefial 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 acet-amidase 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.