结核杆菌CFP-10蛋白的原核表达、纯化及抗血清制备

Prokaryotic expression and purification of CFP-10 of mycobacterium tuberculosis and preparation of its antiserum

目的:获得原核表达及纯化结核杆菌CFP-10蛋白及其抗血清。方法:从H37RV结核杆菌基因组中扩增得到CFP-10的编码基因,通过原核表达系统(BL21-pET28a+)表达及镍亲和层析和Q sepharose阴离子交换层析获得内毒素合格的CFP-10蛋白。将此蛋白免疫家兔,获得抗CFP-10的抗血清,并通过间接ELISA的方法检测其抗体滴度。结果:重组质粒pET28a-CFP-10构建成功,其编码的蛋白在BL21中获得了高效表达,且经纯化获得了内毒素合格的CFP-10蛋白,免疫家兔获得的抗血清效价能达到1∶256 000。结论:成功的表达及纯化了CFP-10蛋白,并制备了高滴度的CFP-10抗血清,为临床血清学检测以及新型结核疫苗研究奠定基础。

Objective：To obtain the CFP-10 protein of mycobacterium tuberculosis by prokaryotic expression and purification,and to prepare the rabbit antiserum.Methods：The CFP-10 gene was amplified from the genome of M.tuberculosis,and then was constructed into the prokaryotic expression vector pET28a.The CFP-10 protein with His-tag was induced expression in E.coli BL21（DE3）,and purified by nickel affinity chromatography,with Q sepharose chromatography to remove endotoxin.The CFP-10 protein was used to immune the rabbit to obtain the polyclonal antibody,which titer has been analyzed by indirect ELISA.Results：The recombinant plasmid pET28a-CFP-10 was constructed successfully,which encoding protein was expressed with high levels and purified with qualified content of endotoxin,and the antiserum of CFP-10 was obtained with high titers.Conclusion：The successful expression and purification of the CFP-10 and the obtained polyclonal antibodies will be very helpful for the development the clinical serologic diagnosis and new anti-tuberculosis vaccine.