番鸭呼肠孤病毒ZJ99株σC基因在甲醇酵母中的表达  

Expression of the Muscovy duck reovirus ZJ99 σC gene in Pichia Pastoris

作　　者：余旭平[1] 刘慧芳[1] 何世成 郑新添[1] 沈琴芳[1] 张秀亮[1] 刘江梅[1] 

机构地区：[1]浙江大学动物科学学院,浙江杭州310029 [2]湖南省兽医总站,湖南长沙410007

出　　处：《黑龙江畜牧兽医》2006年第12期15-18,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：浙江省科技计划重点项目(011102120);宁波市科技计划项目(2002D40015)

摘　　要：将番鸭呼肠孤病毒ZJ99株σC基因插入到酵母表达载体pPIC9K内,转化大肠杆菌TG1,挑取阳性克隆,获得酵母表达重组质粒pPIC9K-σC。用DraⅠ酶切线性化重组表达质粒pPIC9K-σC和对照质粒pPIC9K,电转化至甲醇酵母GS115感受态细胞内,经G418抗性筛选和PCR鉴定,获得了2株pPIC9K-σC酵母阳性整合子和2株pPIC9K空载体对照酵母阳性整合子。分别挑取1株进行甲醇诱导表达,SDS-PAGE检测发现pPIC9K-σC酵母整合子在不同时段的诱导表达上清液中均有可见的目的蛋白条带,分子质量与预计大小相符,而对照pPIC9K酵母整合子的诱导表达上清液中没有相应的蛋白条带。说明番鸭呼肠孤病毒ZJ99株σC基因真核甲醇酵母表达体系构建成功,目的σC蛋白在酵母中能够有效地分泌表达。The σC gene of muscovy duck reovirus （mDRV） ZJ99 strain was subcloned into pP1C9K, the expression vector of Piehia Pastoris anti transformed into E coli TG1, The recombinatant expression plasmid pP1CgK -σC was isolated. The pP1CgK -σC and the control plasmid pP1C9K was then linearized by Dra I and transformed into Pichia Pastoris GS115 strain by electroporation, respectively. Two pP1CgK -σC positive recombinants and two pP1CgK control positive recombinants were isolated by G418 resistance selection and PCR detection. One of them, respectively, was further selected for expression by methanol induction. SDS - PAGE analysis showed that the target protein was expressed in the supernatant of the pP1C9K - σC transformant, but not in that of the pP1CgK. It suggested that the eukaryotic yeast expression system of mDRVσC gone of ZJ99 strain was constructed, and the σC protein can he expressed and secreted into the medium.

关 键 词：番鸭 呼肠孤病毒 σC蛋白 甲醇酵母 真核表达 

分 类 号：S852.659.4[农业科学—基础兽医学]