CFP10-ESAT-6-MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定

目的：在杆状病毒昆虫细胞表达系统（baculovirus expression vector system，BEVS）中表达结核分枝杆菌蛋白CFP10-ESAT-6-MPB64，并鉴定其免疫原性。方法：目的基因CFP10-ESAT-6-MPB64连接到pFastBac转移载体并转化DH10Bac感受态，通过Tn7转座片段将目的基因转座到Bacmid中，得到Bacmid-CFP10-ESAT-6-MPB64穿梭载体，脂质体包被后转染Spodoptera frugiperda（Sf9）细胞收获病毒，病毒转染细胞后收集上清通过Co亲和层析纯化得到目的蛋白。纯化的蛋白免疫Balb/c小鼠并检测血清中特异性抗体滴度及PPD抗体，ELISA检测CFP10-ESAT-6-MPB64蛋白刺激脾脏细胞产生IFN-γ的浓度，MTT法检测目的蛋白对免疫后小鼠脾脏细胞的增殖作用。结果：CFP10-ESAT-6-MPB64在昆虫细胞中成功表达，纯化后蛋白纯度达90%以上，蛋白产量为42mg/L，纯化蛋白能有效刺激Balb/c小鼠产生抗体，提高小鼠脾脏细胞培养基中IFN-γ的含量，目的蛋白在1~50μg/ml之间对脾脏细胞有明显的促增殖作用。

Expression,Purification and Immunogenicity Analysis of Recombinant CFP10-ESAT-6-MPB64 Using the Baculovirus Expression System

Objective: To express Mycobacterium tuberculosis protein CFP10-ESAT-6-MPB64 in baculovirus insect cell expression system, and identify its immunogenicity. Methods: The target gene CFP10-ESAT-6-MPB64 was connected to pFastBac vector, then the pFastBac-CFP10-ESAT-6-MPB64 plasmid which was harvested would transformed to DH10Bac competent, and the target gene was transposition into Bacmid by Tn7 transposase fragment, therefore Bacmid-CFP10-ESAT-6-MPB64 Shuttle vector was obtained. The shuttle vector was packaged by liposomes and transfected Sf9 cells to harvest P1-generation virus, then high titers of P4 generation virus was harvested by repeat transfected Sf9 cells three times. The target protein CFP10-ESAT-6-MPB64 was purified from the supernatant by Co affine chromatography, which were used to immunize Balb/c mice. Antibody changes in serum would be detected, and the proliferation of immunized mice spleen cells would be detected by MTT,detected the IFN-γ secretion by CFP10-ESAT-6-MPB64 stimulated spleen cells by ELISA method. Result: CFP10-ESAT-6-MPB64 successfully expressed in insect cells. The purity of target protein is over 90% and yield up to 42mg/L after purification. Purified protein can effectively stimulate Balb/c mice to produce antibodies, increase the content of IFN-γ medium in mice spleen cells, and significantly promoting proliferation in spleen cells between 1~50μg/ml. Conclusion: CFP10-ESAT-6-MPB64 which has immunogenicity was successfully expressed in baculovirus insect cell expression system, that open a new avenue for tuberculosis vaccine production.