重组人细胞色素P450 2C9在毕赤酵母中的胞内表达  

Intracellular Expression of Human Cytochrome P450 2C9 in Pichia pastoris

作　　者：吴云华[1] 孙鹏宇[1] 

机构地区：[1]中南民族大学生命科学学院国家民委生物技术重点实验室,武汉430074

出　　处：《中南民族大学学报（自然科学版）》2013年第2期41-45,共5页Journal of South-Central University for Nationalities：Natural Science Edition

基　　金：中南民族大学中央高校基金资助项目(CZZ11004)

摘　　要：以含有CYP2C9基因完全编码框的克隆载体质粒DNA为模板,并在人细胞色素P4502C9的5'端插入kozak序列及3'端6×His标签进行PCR扩增,克隆至真核表达载体pPIC3.5K,利用电激法将经Sal I线性化的重组质粒pPIC3.5K-2C9转化至毕赤酵母GS115中,通过抗性、表型和PCR筛选多拷贝整合株,甲醇诱导毕赤酵母GS115/pPIC3.5K-2C9的表达,SDS-PAGE和Western blotting分析鉴定表达产物.结果表明:含有重组质粒的重组pPIC3.5K-2C9表达了大小为55KD的目的蛋白,与预期大小相符,说明人细胞色素CYP2C9可在毕赤酵母的胞内表达.Using cloning vector plasmid DNA containing CYP2C9 complete gene coding frame as a template, the coding region of human cytochrome P450 2C9 with addition of kozak sequence in 5＇-end and 6 ~ His in 3＇-end was cloned into the expression vector pPIC3.5K. The recombinant plasmid pPIC3.5K-2C9 was linearized by Sal I and transformed into Pichia pastori~ GSll5 by electroporation. The insert clones containing multiple copies were selected with geneticin,phenotype and PCR. The expression of Pichia pastoris GS115/pPIC3.5K-2C9 was induced with methanol and analyzed by SDS-PAGE and Western blotting. The results indicated that the recombinant containing recombinant plasmid pPIC3.5K-2C9 expressed an Mw 55KD protein, consistent with the expected value. It was concluded that the human CYP2C9 gene could be expressed intracellularly in Pichia pastoris.

关 键 词：人细胞色素P4502C9 毕赤酵母 胞内表达 

分 类 号：TQ464.7[化学工程—制药化工] Q78[生物学—分子生物学]