鸡PrP基因真核表达载体的构建及其在毕赤酵母GS115中的表达  

Construction of chicken PrP gene eukaryotic expression vector and its expression in Pichia pastoris GS115

作　　者：张天亮[1] 吴润[1] 杨润霞[1] 魏姣[1] 万学瑞[1] 刘霞[2] 刘磊[1] 张小丽[1] 

机构地区：[1]甘肃农业大学动物医学学院,甘肃兰州730070 [2]甘肃农业大学生命科学技术学院,甘肃兰州730070

出　　处：《中国兽医科学》2015年第7期734-739,共6页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31160510);甘肃省自然科学研究基金计划项目(1107RJZA198)

摘　　要：为构建鸡PrP基因的真核表达载体,并通过毕赤酵母表达系统获得鸡朊蛋白（ChPrP）,根据毕赤酵母GS115的密码子偏好性、G＋C含量等特点对目的基因PrP（25～248）进行密码子优化与基因合成,同时以特异性引物扩增获得天然未经密码子优化的目的片段,之后将两种目的片段分别连接于pPIC9K表达载体并转化至毕赤酵母GS115中,经PCR,选择培养基MM、MD方法鉴定表型后以甲醇进行诱导获得目的蛋白,最后以SDS-PAGE检测蛋白表达。结果显示,经PCR及双酶切鉴定,成功构建了真核表达重组载体pPIC9K-PrP（25～248）,并在毕赤酵母GS115中获得鸡朊蛋白,其与未经密码子优化的目的蛋白相比,具有更高的表达量。本研究通过毕赤酵母表达系统成功获得了鸡朊蛋白,为后续研究奠定了基础,也通过密码子优化前后蛋白表达量比对,再一次证实毕赤酵母具有较高的密码子偏好性。The study aimed to construct a chicken prion protein（ChPrP） gene＇s eukaryotic expression vector,and chicken PrP was got through Pichia pastoris expression system. According to the codon prefe- rences, G＋C content and other characteristics of P. pastoris GS115, the codon of target g ene PrP（25- 248） was optimized and synthesized,at the same time, the natural and without codon-optimized target fragment was amplified with specific primers. Afterwards,two target fragments were cloned into pPIC9K expression vector and transformed into P. pastoris GSl15. After phenotypic identification with both PCR and choose medium MM/MD methods, the target proteins were induced by methanol. Finally, the protein expression of PrP was detected by SDS-PAGE. In result,we successfully constructed recombinant eukaryotic expression vector pPIC9K-PrP（25--248） by identifying by PCR and double enzyme methods,and got chicken PrP in P. pastoris GS115. Compared with without codon-optimized target protein,it has higher expression. In con- clusion,this study has successfully got chicken PrP by P. pastoris expression system, which laid the foun- dation for subsequent research. In addition,it was again confirmed that Pichia has high codon preferences through compared with quantity of protein expression between before and after codon-optimization.

关 键 词：鸡朊蛋白 PPIC9K 毕赤酵母GS115 真核表达 

分 类 号：S852.659.7[农业科学—基础兽医学]