Wang Xiao-ying, Bao Lang, Zhao Ming-cai, Zhang Hui-dong, Long Yang
Research Unit of Infection and Immunity, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2006 May;37(3):353-6.
To express a recombinant fusion protein CFP10-ESAT6 of Mycobacterium tuberculosis, and obtain the polyclonal antibodies of this fusion protein by immune rabbit.
The 630 bp cfpl0-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.
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.
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.
表达结核分枝杆菌重组融合蛋白CFP10-ESAT6，并通过免疫兔获得该融合蛋白的多克隆抗体。
从结核分枝杆菌标准菌株H37Rv的基因组DNA中扩增出630 bp的cfpl0-esat6融合基因片段，插入表达质粒pET32a(+)中，构建重组质粒pET-cfp10-esat6。将重组表达质粒转化至大肠杆菌BL21(DE3)。经IPTG诱导后表达出带有His标签的融合蛋白CFP10-ESAT6，并用亲和层析法进行纯化。用该蛋白免疫兔获得多克隆抗体，并进行Western-blot和ELISA分析。
成功构建了重组质粒pET-cfp10-esat6，重组融合蛋白CFP10-ESAT6能较高水平表达，并获得了融合蛋白的多克隆抗体。
重组融合蛋白CFP10-ESAT6的成功构建与表达以及获得的多克隆抗体，将对新型抗结核疫苗的研发及临床血清学诊断有很大帮助。
