结核分枝杆菌CFP10-ESAT6融合蛋白的高效表达及免疫原性研究

Expression of the Fusion Protein CFP10-ESAT6 of Mycobacterium Tuberculosis and the Study of Its Immunogenicity

目的高效表达结核分枝杆菌6kDa早期分泌性抗原靶(6kDaearlysecretoryantigenictarget,ESAT6)和培养滤液蛋白10(culturefiltrateprotein10,CFP10)的融合蛋白,通过免疫动物,获得CFP10-ESAT6融合蛋白的多克隆抗体。方法以结核分枝杆菌标准株H37Rv基因组DNA为模板,扩增cfp10-esat6融合基因,将其与表达质粒pET32a(+)构建成重组质粒pET-cfp10-esat6。在大肠杆菌BL21(DE3)中表达带组氨酸标签的融合蛋白,经亲和层析得到纯化CFP10-ESAT6蛋白。用该蛋白免疫成年家兔,获得的抗CFP10-ESAT6融合蛋白的多克隆抗体,作Western-blot和ELISA分析。结果重组质粒pET-cfp10-esat6构建成功,融合蛋白CFP10-ESAT6大量表达,多克隆抗体制备成功(1∶106)。结论成功构建并表达了CFP10-ESAT6融合蛋白,制备了大量的CFP10-ESAT6融合蛋白的多克隆抗体,为发展新型结核病疫苗和临床血清学检测奠定了实验基础。

Objective To express a recombinant fusion protein CFP10-ESAT6 of Mycobacterium tuberculosis, and obtain the polyclonal antibodies of this fusion protein by immune rabbit. Methods The 630 bp cfp10-esat6 fusion gene fragments were amplified from the genomic DNA of a Mycobacterium tuberculosis reference strain H37Rv and inserted into the expression plasmid pET32a （W） to generate the recombinant plasmid pETcfp10-esat6. The recombinat expression plasmid was transformed into E. coli BL21 （DE3）. The fused protein CFP10-ESAT6 with His-tag was expressed after inducing with IPTG and purified with affinity chromatography. This protein was used to immune the rabbit to obtained the polyclonal antibodies, and been analyzed with Westernblot and ELISA. Results The recombinant plasmid pET-cfp10-esat6 was success fully constructed, the recombinant fusion protein CFP10-ESAT6 could be expressed at relatively high levels, and the polyclonal antibodies of fusion protein were obtained. Conclusion The successful construction and expression of the recombinant fusion protein CFP10-ESAT6 and the obtained polyclonal antibodies will be very helpful for the development of new anti-tuberculosis vaccine and the clinical serologic diagnosis.