猪带绦虫TSOL18重组耻垢分枝杆菌疫苗的构建及其表达

Construction and expression of a recombinant Mycobacterium smegmatis vaccine containing Taenia solium TSOL18

目的构建猪带绦虫TSOL18重组耻垢分枝杆菌疫苗,分析猪带绦虫TSOL18基因在重组耻垢分枝杆菌中的表达情况。方法采用PCR方法从重组质粒pGEX-TSOL18中扩增TSOL18基因,克隆入pMV361载体,构建pMV361-TSOL18穿梭质粒,经酶切和测序鉴定后电穿孔法转化入耻垢分枝杆菌,进行单克隆菌落筛选及PCR鉴定。构建的猪带绦虫TSOL18重组耻垢分枝杆菌疫苗经热诱导后,以SDS-PAGE分析及Western blot分析TSOL18基因在重组耻垢分枝杆菌中的表达情况。结果成功扩增出TSOL18目的基因,大小约为393 bp,与预期值相符;经酶切验证,pMV361-TSOL18穿梭质粒构建成功;PCR鉴定猪带绦虫TSOL18重组耻垢分枝杆菌疫苗构建成功。SDS-PAGE电泳检测重组菌表达的目的蛋白TSOL18,相对分子质量为15×10^3与预期相符;Western blot分析该蛋白能被TSOL18兔抗血清识别。结论成功构建了猪带绦虫TSOL18重组耻垢分枝杆菌疫苗,猪带绦虫TSOL18基因在耻垢分枝杆菌中成功表达,表达蛋白具有反应原性,其免疫原性有待进一步研究。

Objective To construct a recombinant Mycobacterium smegmatis vaccine containing TSOL18 of Taenia solium in order to ascertain the expression of T. solium TSOL18 in recombinant M. smegmatis. Methods The TSOL18 gene was amplified from the recombinant plasmid pGEX-TSOL18 using PCR and cloned into the vector pMV361 to construct a pMV361-TSOL18 shuttle plasmid. After digestion and identification, the shuttle plasmid was transformed into M. smegmatis via electroporation. After the monoclonal colonies were screened and identified using PCR, a recombinant M. smegmatis vaccine containing T. solium TSOL18 was constructed. After heat induction, SDS-PAGE and Western blotting were used to analyze and identify the expression of the TSOL18 gene in M. smegmatis. Results PCR indicated that the TSOL18 gene(393 bp in length) was successfully amplified. The shuttle plasmid pMV361-TSOL18 was successfully constructed using enzyme digestion. A recombinant M. smegmatis vaccine containing T. solium TSOL18 was successfully constructed according to PCR. After heat induction, the target protein TSOL18 was detected using SDS-PAGE. Its relative molecular mass was 15×10^3, which was consistent with the expected value. Western blotting indicated that the protein was recognized by TSOL18 rabbit antiserum. Conclusion A recombinant M. smegmatis vaccine containing T. solium TSOL18 was successfully constructed. Recombinant TSOL18 protein was successfully expressed in M. smegmatis. The recombinant TSOL18 protein is immunoreactive, though its immunogenicity needs to be studied further. This finding has laid the foundation for further study of M. smegmatis vaccines.