HIV-1中国株CN54 Gag蛋白在毕赤酵母中的表达纯化和鉴定  

Expression in Pichia pastoris, fermentation and purification of HIV-1 CN54 Gag antigen

作　　者：郝彦玲[1] 马民强[1] 傅晶晶[1] 李海山[1] 王贻杰[2] 刘颖[1] 吴岚[1] 刘勇[1] 邵一鸣 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京100050 [2]福建师范大学生物工程学院

出　　处：《中华实验和临床病毒学杂志》2005年第2期128-131,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：国家高技术研究发展计划 (863计划 )资助项目(2 0 0 1AA2 15 0 3 1) ;科技部国际合作重大课题资助项目 (HIV蛋白工程疫苗及乙型肝炎预防控制新方法的研究 )

摘　　要：目的　利用毕赤酵母表达系统高效表达HIV 1中国株CN5 4的Gag蛋白,初步确立发酵和纯化工艺。方法　PCR扩增CN5 4Gag基因,插入毕赤酵母表达载体pPS1 0 ,电转化毕赤酵母菌株GS115 ,G4 18筛选高表达工程菌,利用5L发酵罐进行高密发酵,通过SPSepharoseFF和DEAESeparoseFF柱层析,用HIV 1阳性血清和p2 4抗原检测试剂盒对纯化后的Gag蛋白进行免疫学性质的鉴定。结果　构建了高效表达CN5 4Gag蛋白的毕赤酵母工程菌株,发酵罐培养的菌体密度吸光度(A6 0 0 值)超过30 0 ,Gag蛋白表达量达到12 0mg L。纯化后的Gag蛋白纯度高于90 % ,p2 4检测强阳性并能够与HIV 1阳性血清发生特异的抗原抗体结合反应。结论　本研究在毕赤酵母中高效表达了HIV 1中国株CN5 4的Gag蛋白,并进行了纯化和鉴定,为针对我国新一代HIV蛋白亚单位疫苗的研制奠定了基础。Objective To express the Gag protein of HIV-1 strain CN54 in Pichia pastoris (P.pastoris), optimize fermentation parameters and purify Gag antigen. Methods The Gag gene was subcloned into downstream of aox1 promoter of Pichia expression vector pPS1.0, an integrative vector which possesses an identical 5' untranslated region as the natural aox1 gene and employs both in vitro construction and in vivo selection for multi-copy integrants. The recombinant vector was introduced into P.pastoris strain GS115 by electroporation and selected with G418 for Gag gene integration. Super G418 resistant clones were selected and screened for Gag expression. The engineered P.pastoris was cultured to high cell density (>300 A 600 Units/ml) in a 5L fermentor. Through methanol induction, the expression level of Gag reached 120 mg/L. Intracellularly expressed Gag was released by high-pressure homogenization and purified through Sepharose FF and DEAE Sepharose FF column chromatography, the purity of Gag reached up to 90%. Results Western-blotting suggested that purified Gag expressed in P.pastoris could react specifically with serum from HIV infected individual. Conclusion Gag antigen expressed in P.pastoris has provided a good basis for the development of a new generation of HIV vaccine candidates against some Chinese prevalent strains.

关 键 词：GAG蛋白 HIV-1 中国株 鉴定 HIV蛋白亚单位疫苗 SEPHAROSE 毕赤酵母表达系统 抗原抗体结合反应 抗原检测试剂盒 高效表达 酵母表达载体 阳性血清 GAG基因 PCR扩增 GS115 免疫学性质 纯化工艺 DEAE 工程菌株 菌体密度 蛋白纯度 

分 类 号：TQ464[化学工程—制药化工]