乙型肝炎病毒全长PreS蛋白基因在酵母中的表达  

Expression of the Full Length preS Gene of Hepatitis B virus in Pichia pastoris

作　　者：孙明颢[1] 叶林伯[1] 郜金荣[1] 贺石汉[1] 吴正辉[1] 

机构地区：[1]武汉大学生命科学院病毒研究所,湖北武汉430072

出　　处：《中国病毒学》2003年第2期99-103,共5页Virologica Sinica

基　　金：武汉市攻关项目(20026002091)

摘　　要：应用直接提取法从武汉地区乙型肝炎病人患者阳性血清中提取HBV基因组,以此作为模板,用引物PCR方法获得全长preS基因,该片段的DNA大小1 203bp.克隆到pUCm-T载体中,应用M13通用引物进行序列测定,与中国HBV标准株序列比较,证实基因型为Adr亚型。有24个核苷酸不同,preS基因包含3个起始密码ATG分别为preS1,preS2,和S的翻译起点。然后将preS基因克隆到毕赤酵母表达载体pPIC9K中,将Sal I线性化的pPIC9K-preS质粒用电击法导入巴斯德—毕赤酵母GS115His-中。通过MD-G418平板筛选和5AOX1和3AOX1引物PCR鉴定获得稳定重组HBV全长preS基因的His+Mut+酵母工程菌株。此酵母菌株在合适的培养条件和甲醇诱导下高效表达产生全长PreS蛋白并可分泌到培养液中,SDS-PAGE显示培养液中含有分子量约为48kDa的带;微孔ELISIA法证实表达并分泌到培养液中的PreS蛋白能够与HBV抗体阳性血清发生特异性反应。Hepatiti B virus (HBV) genome DNA was directly extracted from human serum infected with HBV.The full length preS gene DNA was obtained by PCR using the HBV genome DNA as template. Then it was cloned into the pUCm-T vector and sequenced using the M13 Primers. The sequencing data shows that it contains 1 203bp and it length with 24 different nucleotides compared with the standard sequence of HBV (subtype adr) in China, it was confirmed to be the preS gene with three ATGs which corresponded to the initiation codon of PrsSl, PreS2 and S proteins respectively. This DNA fragment was cloned into the SnaB l-AvrⅡ sites of expressing vector pPIC9K, in framed with the AOXI promotor and then the pPIC9K-PreS recombined plasmid DNA linearized by Sal I was introduc- ed to Pichia pastoris GSll5 by electroporation device. GSll5-pPIC9K-PreS was got by selecting with MD-G418 plates and identifying with PCR. The GSll5-pPIC9K-PreS can grow in the media with methanol and can produce the PreS protein in secreted form with molecular weight 48KD as detected by SDS-PAGE. ELISA experiment proved that the protein can react with the positive human serum against HBV.

关 键 词：乙型肝炎病毒 全长PreS蛋白基因 ADR亚型 疫苗 毕赤酵母表达系统 

分 类 号：R392[医药卫生—免疫学]