乙型肝炎病毒表面抗原preS2+S基因在Pichia Pastoris酵母系统的分泌表达  

Secreted Expression of Hepatitis B Virus PreS2+S Gene in Pichia Pasto ris Expression System

作　　者：徐丽宏[1] 梁国栋[1] 付士红[1] 宋宏[1] 王大维[1] 苏乃伦[1] 夏国良[1] 张智清[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京100052

出　　处：《病毒学报》2003年第3期224-229,共6页Chinese Journal of Virology

基　　金：国家十五科技攻关项目(编号:2001BA705B05)

摘　　要：拟获得adw2亚型乙型肝炎(乙肝)病毒表面抗原preS2+S基因在PichiaPastoris酵母分泌型表达系统(pPIC9K)的高效表达。实验首先将adw2亚型乙肝病毒表面抗原preS2+S基因重组到分泌型酵母表达载体(pPIC9K)形成表达质粒,电转化酵母细胞KM71,G418筛选多拷贝整合克隆,经甲醇诱导表达并用SDS-PAGE电泳及酶免疫法检测表达产物。经100个克隆筛选获得了表达量较高的表达菌株WC4。该菌株甲醇诱导后细胞上清10倍浓缩SDS-PAGE电泳检测显示,细胞上清中有特异蛋白条带,且第6天表达量最高,表达产物单体分子量为31kD左右。用美国雅培公司AUZYMEMONOCLONAL试剂盒估算表达量为2μg/100OD600细胞。上述结果表明,乙肝病毒表面抗原preS2+S基因在本系统中获得了分泌表达。同时检测了酵母细胞裂解液中特异蛋白质的表达,结果发现,自甲醇诱导后第一天即可检测到表达产物,而且除了第6天细胞外表达量高于细胞内外,其余各天的表达水平均表现为细胞内高于细胞外。以上结果提示,利用分泌型酵母表达系统表达乙肝病毒表面抗原在技术上可行,但表达产量偏低,一些蛋白滞留在细胞内未能分泌到培养基中。In order to obtain high-l evel secreted expression of hepati tis B virus(HBV) preS2+S gene in P ichia Pastoris system,the HBV surf ace antigen(HBsAg) preS2+S gene wa s cloned into the secreted express ion vector pPIC9K at first.The rec ombinant plasmid p9Kadw2S was then electroporoted into yeast Pichia P astoris cells KM71.Multiinsertion transformants were screened with G 418 resistance.After methanol indu ction,the expressed HBsAg protein were analyzed by SDS-PAGE and ELIS A.The high-level expression clones were selected from 100 positive co lonies.Specific HBsAg protein expr ession could be detected with SDS- PAGE from 10× concentrated culture medium.The peak expression was on the 6th day after methanol inducti on.The molecular weight of the monomer was about 31kD.When the quantities of HBsAg protein were estimated wi th the AUZYME MONOCLONAL kit,WC4 p roduced about 2μg/100 OD600 cells i n the culture medium.All the resul ts above suggest that adw2 subtype preS2+S gene can be specifically e xpressed and secreted.We also meas ured the expressed HBsAg protein i n the cell extract from the 1st da y after induction by methanol.When comparing the expression level in the culture medium and cell extrac t on the same day,we found that e xcept the 6th day,the expression level was higher in the cell extra ct than in the culture medium on a ll the other days after methanol i nduction.This result indicates tha t the proteins can't be secreted i nto the culture medium completely, most of them still retain in yeast cells.

关 键 词：乙型肝炎病毒 表面抗原pres2+S基因 酵母系统 分泌表达 

分 类 号：R373.21[医药卫生—病原生物学] Q786[医药卫生—基础医学]