人载脂蛋白基因apoAⅠ在毕赤氏酵母中的胞内表达  

Intracellular Expression of Human Apolipoprotein AⅠGene in Yeast Pichia pastoris

作　　者：杨婷婷[1] 赵志安[1] 张彦[1] 宋大新[1] 

机构地区：[1]复旦大学生命科学学院,上海200433

出　　处：《复旦学报（自然科学版）》2004年第6期1035-1038,共4页Journal of Fudan University：Natural Science

基　　金：复旦大学Med X基金重点资助项目;上海市科学技术委员会资助项目(024319204)

摘　　要：将PCR扩增得到的apoAⅠ基因片段插入胞内表达质粒pPIC3.5K,重组的pPIC3.5K apoAⅠ用BglⅡ酶切后,转化PichiapastorisGS115菌株,筛选获得G418高抗性转化子,再通过PCR鉴定证实apoAⅠ基因已整合到染色体上.其转化子经摇瓶发酵和甲醇诱导,收集细胞裂解后,用SDS PAGE与Westernblotting检测,证明胞内有明显的rApoAⅠ表达,其分子质量与人血浆提取的ApoAⅠ相同.apoAⅠwas inserted into intracellular expression vectors pPIC3.5K. The recombined pPIC3.5K-apoAⅠwas digested by BglⅡand transformed into Pichia pastoris strain GS115. Then the transformants with high G418 resistance were selected and identified by PCR. After the transformants grew in the shaking flask and was induced by methynol,SDS-PAGE analysis and Western blotting showed that they expressed rApoAⅠ,and the molecular weight is the same as that of natural ApoAⅠprotein purified from human serum.

关 键 词：表达质粒 ApoAⅠ 载脂蛋白 基因 人血浆 再通 诱导 毕赤氏酵母 转化子 细胞裂解 

分 类 号：Q78[生物学—分子生物学] R589.2[医药卫生—内分泌]