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目的构建结核分枝杆菌去标签重组蛋白Mtb8.4-Hspx(MH)和Hspx-Mtb8.4(HM)原核表达载体,并在大肠埃希菌株BL21(DE3)中表达。方法以结核分枝杆菌H37Rv基因组DNA为模板,PCR扩增Mtb8.4、Hspx基因,产物经纯化后与载体PET30a连接、转化及测序鉴定,构建原核重组表达质粒PET30a-Mtb8.4和PET30a-Hspx。抽提重组质粒,产物经纯化后与目的基因Hspx、Mtb8.4连接,转化及测序鉴定,构建重组目的基因质粒PET30a-Mtb8.4-Hspx(PET30a-MH)和PET30a-Hspx-Mtb8.4(PET30a-HM)载体,转化大肠埃希菌BL21,以IPTG诱导,SDS-PAGE电泳检测表达产物。结果 SDS-PAGE蛋白电泳验证去标签MH和HM融合蛋白分子质量单位为28 ku,与预期值相符。结论本研究成功表达去标签结核杆菌重组蛋白MH和HM,为结核亚单位疫苗免疫原性和临床前研究奠定了基础。
Objective To construct a prokaryotic expression vector for Mtb8.4-Hspx (MH) and Hspx-Mtb8.4 (HM) recombinant Mycobacterium tuberculosis and express it in Escherichia coli BL21 (DE3). Methods Mycobacterium tuberculosis H37Rv genomic DNA was used as a template to amplify Mtb8.4 and Hspx genes. The purified product was ligated with vector PET30a, transformed and sequenced. The recombinant prokaryotic expression plasmids PET30a-Mtb8.4 and PET30a-Hspx . The recombinant plasmids were extracted and purified. The products were purified and ligated with the target genes Hspx and Mtb8.4. The recombinant plasmids were transformed and sequenced. The recombinant plasmids of PET30a-Mtb8.4-Hspx (PET30a-MH) and PET30a-Hspx-Mtb8.4 PET30a-HM) vector was transformed into Escherichia coli BL21, induced by IPTG, and the expression product was detected by SDS-PAGE electrophoresis. Results SDS-PAGE electrophoresis showed that the molecular mass units of de-labeled MH and HM fusion proteins were 28 ku, which was consistent with the expected value. Conclusion The recombinant Mycobacterium tuberculosis MH and HM were successfully expressed in this study, which laid the foundation for the immunogenicity and preclinical study of TB subunit vaccine.