结核分枝杆菌CFP10-ESAT6-TB7.7的融合表达及其在小鼠上的细胞免疫学特性(英文)

Fusion expression of CFP10-ESAT6-TB7.7 of Mycobacterium tuberculosis and its cellular immunoproperties in mice

目的构建结核分枝杆菌CFP10-ESAT6-TB7.7融合蛋白的表达质粒,利用大肠杆菌表达系统获得原核蛋白,在小鼠模型上评价其细胞免疫学特性。方法通过PCR扩增出lhp-linker-esat6-linker和linker-Rv2654c片段,构建正确的pET32a(+)-lhp-linker-esat6-linker-Rv2654c原核表达质粒,转化BL21(DE3)后在IPTG诱导下表达,并纯化该融合蛋白,western blot验证蛋白的免疫原性,通过T细胞增殖实验和夹心ELISA评价其细胞免疫学特性。结果成功构建pET32a(+)-lhp-linker-esat6-linker-Rv2654c原核表达质粒,SDS-PAGE显示在45kDa处出现目的条带,Western blot证明融合蛋白对anti-ESAT6,anti-CFP10,anti-His单抗和rGST-TB7.7多抗血清均有反应,说明该融合蛋白具有良好的免疫反应性,T淋巴细胞增殖实验显示该蛋白能明显引起小鼠的T细胞免疫反应。夹心ELISA实验也表明,融合蛋白刺激产生的IFN-γ和IL-4水平与对照组相比均有显著性差异,同时融合蛋白诱导产生的IFN-γ水平明显高于IL-4。结论 CFP10-ESAT6-TB7.7融合蛋白在大肠杆菌系统中实现可溶性表达,并获得免疫反应性良好的纯化产物,该融合蛋白能引起明显的T细胞增殖,具有诱导Th1型免疫应答的特性。

ABSTRACT..CFP10-ESAT6-TB7.7 fusion protein of Mycobacterium tuberculosis was expressed in E. coli BL21（DE3） and its cellular immunoproperties in murine model were analysed. Recombinant plasmid pET32a （＋）-lhp-linker-esat6-1inker- Rv2654c was constructed and induced with IPTG after transforming into BL21 （DE3）. SDS-PAGE displayed the high level expression of the fusion protein while Western blot further confirmed its good immunoreactivity with different antibodies. Then, T lymphocyte proliferation assay and sandwich ELISA were used to evaluate the cellular immunoproperties of the fusion protein in mouse. Significant T cell proliferation was observed from the splenocytes of rHis-CFP10-ESAT6-TBT. 7 immunized group after stimulation with the mixture of rGST-CFP10, rGST-ESAT6 and rGST-TB7. 7 proteins. Furthermore, higher IFN-;~ specific secreting level rather than IL-4 were detected by sandwich ELISA assay from splenic ceils of C57BL/6 mice which immunized with the fusion protein. These results showed that rHis-CFP10-ESAT6-TBT. 7 fusion protein was expressed successful with the ability to induce Thl cell mediated cellular immune responses.