SARS冠状病毒M基因的克隆及其表达  

Cloning and Expression of SARS Coronavirus M Gene

作　　者：晏辉钧[1] 赵卫[2] 方丹云[1] 周经姣[1] 龙北国[2] 张文炳[2] 郭辉玉[1] 江丽芳[1] 

机构地区：[1]中山大学基础医学院微生物学教研室,广东广州510080 [2]南方医科大学热带卫生学系微生物学教研室,广东广州510515

出　　处：《中国病毒学》2005年第1期1-4,共4页Virologica Sinica

基　　金：国家自然科学基金(编号:30340013);广东省科技攻关项目(编号:532014202028)

摘　　要：从SARS冠状病毒(GD322株)组织培养上清中提取 RNA,进行 RT PCR扩增其 M基因并克隆到巴氏毕赤酵母表达载体 pPICZαB,电穿孔法将重组体整合入酵母菌 P. pastoris,经抗生素 Zeocin筛选产生的转化子进行表型鉴定,取Mut+菌用甲醇诱导表达,SDS PAGE检测其表达产物。Mut+ 酵母转化菌经甲醇诱导可分泌表达约 65kDa和42 kDa的蛋白质,与SARS恢复期病人血清的免疫印迹证实它们为特异的重组M蛋白质,且获得的重组M蛋白质具有免疫反应性。The full-length gene of membrane protein ( M )of SARS coronavirus (SARS-CoV) was cloned using RT-PCR from the Vero-E6 cells infected with SARS-CoV (GD322 strain) and inserted into the multi-cloning site of the pPICZαB vector. The recombinant plasmid was transtormed into P. pastoris strain X-33 by electroporation and selected by Zeocin. Mut phenotype determination was performed on the yeast transformants and then expression of Mut^+ colonies were induced by methanol. The expressed products were analyzed by SDS-PAGE and Western blotting. Secreted expression was performed by screening Mut^+ colonies in the yeast (transformants). The molecular mass of the recombinant M protein was approximately 65 kDa and 42 kDa and secreted into culture medium when induced with methanol. The expressed protein was able to (react) with SARS convalescence polyclonal antibody.

关 键 词：SARS冠状病毒 M基因 克隆 巴氏毕赤酵母 表达 

分 类 号：R346[医药卫生—基础医学]