摘要
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1-50g/ml之间对脾脏细胞有明显的促增殖作用。
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 Pl-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 supematant 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 MTF, 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-7 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.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2014年第6期23-30,共8页
China Biotechnology
基金
国家"863"计划(SQ2011AA100606)
浙江省自然科学基金(LY13H300006)
2012年省级重点科技创新团队项目(2012R10042-10)
2012年吉林省高层次创新创业引进人才项目资助项目