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SalI线性化的 pPIC9K-preS质粒用电击法导入巴斯德一毕赤酵母GS115His冲。通过MD．G418平板筛选和5’AOX1和3’AOX1 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I-IB~抗体阳性血清发生特异性反应。

Hepatitf B virus(HBV1 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 w0s cloned into the pUCm—T vector and sequenced using the M 1 3 Primers．The sequencing data shows that it contains 1 203bp an d 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 PrsS1，PreS2 an d S proteins respectively．Th is DNA fragment was cloned into the SnaB 1-Avr II sites of expressing vector pPIC9K，in framed with the AOXI promotor and then the pPIC9K—PreS recombined plasmid DNA linearized by Sal 1 was introduc‘ ed to Pichia pastoris GSll5 by electroporation device．GSll5一pPIC9K—PreS was got by selecting with MD-G418 plates an d identifying with PCR．Th e GSll5-pPIC9K,PreS Can grow in the media with methan ol 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