结核分枝杆菌CFP10-ESAT6融合蛋白的克隆表达及效价测定

目的在大肠杆菌中表达结核分枝杆菌RD1区CFP10-ESAT6融合蛋白,测定融合蛋白对结核分枝杆菌致敏豚鼠的效价。方法以结核分枝杆菌标准株H₃₇R_v为模板,采用Overlap PCR方法扩增CFP10-ESAT6融合基因,并克隆入pET-30a质粒,IPTG诱导工程菌表达融合蛋白,离子层析柱分离纯化融合蛋白。以结核分枝杆菌致敏豚鼠进行皮肤变态反应(DTH)测定。结果结核分枝杆菌融合蛋白在大肠杆菌中以可溶形式表达,融合蛋白占总菌体蛋白的30%以上,经离子层析柱纯化纯度达95%以上。重组蛋白可诱导结核分枝杆菌致敏豚鼠产生迟发型超敏反应,2.5μg/ml重组蛋白诱导豚鼠皮试反应与TB-PPD(50 IU/ml)等效。结论重组融合蛋白有望成为结核感染皮试诊断用新试剂。

Clone, expression in E.coli and efficiency measurement of recombinant CFP10-ESAT6 fusion proteins from Mycobacterium turberculosis

Objective The recombinant CFP10-ESAT6 fusion protein coding by the genes located in RD1 regions of genome of Mycobacterium tuberculosis was expressed in E.coli,and its skin reactions were detected inguinea pigs. Methods A genomic DNA of Mycobacterium tuberculosis H37Rv strain was used as template,amplified the cfp10-esat6 fusion gene by Overlap PCR.PCR production was cloned into pET-30a plasmid.The recombinant E.coli was induced by IPTG.The supernatant liquid of centrifugal ultrasonic bacteria was purified through anion column.Skin reactions of fusion protein were tested in guinea pigs infected with M.tuberculosis. Results The fusion proteins was expressed as soluble state in E.coli.The content of recombinant CFP10-ESAT6 fusion proteins was more than 30% of total proteins,and more than 95% purity was acquired through anion column.The fusion proteins could induce DTH in guinea pigs infected with M.tuberculosis and 2.5 μg/ml recombinant proteins induced the same DTH as PPD（50 IU/ml）. Conclusion The recombinant CFP10-ESAT6 fusion proteins is hopeful to become new skin test reagent in the differential diagnosis of tuberculosis.