PRRSV河南株ORF7基因的克隆表达及条件优化  

Cloning and Prokaryotic Expression of the ORF7 Gene of PRRSV Henan Strain

作　　者：刘明[1,2] 张改平[2] 肖治军[2] 郭军庆[2] 乔松林[2] 王丽[2] 史西保[2] 刘运超[1,2] 游雷鸣[1,2] 王爱萍[1] 

机构地区：[1]郑州大学生物工程系,河南郑州450001 [2]河南省农业科学院,农业部动物免疫学重点实验室/河南省动物免疫学重点实验室,河南郑州450002

出　　处：《河南农业科学》2011年第1期136-140,共5页Journal of Henan Agricultural Sciences

基　　金：国家自然科学基金项目(30730068)

摘　　要：从猪繁殖与呼吸综合症病毒(PRRSV)河南分离株(HN07-1、HN07-2)的细胞培养物中提取RNA,根据参考株IAF-exp91 N蛋白基因序列设计出2对引物,成功克隆出ORF7全长基因,并连接至pMD-19T载体。基因测序结果表明,2株PRRSV河南分离株ORF7基因序列完全相同;该序列与HB-4(hs)同源性为100%,与标准美洲株VR-2332和我国早期分离的BJ-4株同源性为93.8%。将该基因亚克隆到原核表达载体pGEX-6p-1后,转化大肠杆菌BL21,IPTG诱导表达并对表达条件进行优化。SDS-PAGE和Western-blot结果表明,成功表达了约40 kD的融合蛋白;使用1.0 mmol/L的IPTG在2×YT培养基中诱导8 h可获得最大表达量。Total RNA was extracted from the cell cultures of two Henan strains of porcine reproductive and respiration syndrome virus（PRRSV）,HN07-1 and HN07-2,and the ORF7 gene of PRRSV was amplified by RT-PCR using specific primers designed according to the sequence of the nucleocapsid protein（N） gene of IAF-exp91.The PCR products were connected into pMD-19T vector,cloned and sequenced.The results showed that the ORF7 genes of the two strains were same to each other,and the homology of ORF7 gene with HB-4（hs）,VR-2332,and BJ-4 was 100%,93.8%,and 93.8%,respectively.A prokaryotic expression vector pGEX-6p-1 containing ORF7 gene was constructed,transformed into E.coli BL21 and induced with IPTG.SDS-PAGE and Western-blot confirmed that the recombinant protein of 40kD was successfully expressed in E.coli BL21.

关 键 词：猪繁殖与呼吸综合征 反转录 克隆 表达 WESTERN-BLOT 美洲型 

分 类 号：S852.65[农业科学—基础兽医学]