高致病性猪繁殖与呼吸综合征病毒TA-12株ORF6基因的克隆、鉴定及真核表达  

Cloning,Identification and Eukaryotic Expression of ORF6 Gene from Highly Pathogenic PRRSV Strain TA-12

作　　者：王彤彤[1] 汪晓飞[1] 李心安 田国宁[2] 刘立涛[1] 王广文[1] 李宁[1] 王方昆[1] 李红梅[1] 赵孝民[1] 肖一红[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271018 [2]潍坊出入境检验检疫局,潍坊261041

出　　处：《生物技术通报》2013年第11期159-163,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30901063)

摘　　要：旨在获得HP-PRRSV TA-12(GenBank登录号:HQ416720)分离株ORF6蛋白的高效表达,将HP-PRRSV TA-12分离株的ORF6基因克隆到载体PfastBac HTB,并经转座进入E.coli含有杆状病毒质粒Bacmid的DH10BacTM细胞株,通过蓝白斑筛选和PCR鉴定出阳性重组杆粒,再将提取的重组杆粒Bacmid-ORF6转染昆虫细胞sf9,使其表达ORF6蛋白。经Western blot鉴定,在感染重组杆状病毒后第4天ORF6蛋白表达量最大,达到0.05μg/μL。表达的ORF6蛋白主要存在于细胞质中。To obtain the efficient expression of ORF6 proteins of HP-PRRSV strain TA-12, the eukaryotic expression plasmid PfastBac HTB-ORF6 was constructed and transformed into DH10BacTM to get Bacmid-ORF6. The recombined plasmid was screened and identified by blue-white color selection and PCR using specific primers. Then the Bacmid-ORF6 was transfected into sf9 cells for expression. The results showed that the protein was expressed and the highest expression amount of 0.05 μg/μL was found at four days post infection using Western blot, and the expressed protein was mainly located in cytoplasm. The successful expression of the ORF6 protein has laid good foundation for the study of its potential function in the process of PRRSV infection.

关 键 词：高致病性猪繁殖与呼吸综合征病毒 ORF6蛋白 真核表达 

分 类 号：S852.65[农业科学—基础兽医学]