结核分枝杆菌CFP10-ESAT6融合蛋白的高效表达及免疫原性研究

目的高效表达结核分枝杆菌6kDa早期分泌性抗原靶（6kDa early secretory antigenic target，ESAT6）和培养滤液蛋白10（culture filtrate protein 10，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（1：10^4）。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。

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

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(+) to generate the recombinant plasmid pET-cfp10-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 Western-blot 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.