猪繁殖与呼吸综合征病毒ORF5基因原核表达载体的构建与鉴定  

Construction and Identification of PRRSV ORF5 Gene Prokaryotic Expression Vector

作　　者：周玉照 马志亮[1] 孙亭亭[1] 杨洁[1] 张小苗[1] 柴俊[1] 张娜娜[1] 张以芳[1] 

机构地区：[1]云南农业大学动物科学技术学院,昆明650201

出　　处：《中国畜牧兽医》2015年第11期2880-2887,共8页China Animal Husbandry & Veterinary Medicine

基　　金：云南省重点新产品开发(2014BB04);云南省院士专家工作站建设项目(2014IC034)

摘　　要：本试验旨在构建能表达猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因的重组质粒。用疑似患猪繁殖与呼吸综合征(PRRS)病猪的脾脏、肺脏病料提取RNA,根据PRRSVORF5基因设计1对引物,进行RT-PCR扩增,将ORF5目的基因克隆到pMD19-T载体中,得到pMD19-ORF5重组菌。根据测序结果和表达载体特点,设计1对带有NcoⅠ和XbaⅠ酶切位点的引物,扩增目的片段(dORF5),将其分别与pProEXHTb载体、pNZ8149载体连接,得到HTb-dORF5/DE3和pNZ8149-dORF5/NZ3900重组菌,分别用IPTG和Nisin诱导,再进行SDS-PAGE和Western blotting分析。结果显示,HTb-dORF5/DE3重组菌在1.5mmol/L IPTG诱导时表达量最高,通过SDSPAGE和Western blotting分析,在约22ku处有特异性条带,且具有反应原性;pNZ8149-dORF5/NZ3900重组菌在20ng/mL Nisin诱导时表达量最高,通过SDS-PAGE和Western blotting分析,在约19ku处有特异性条带,间接免疫荧光试验有特异性绿色荧光。本研究成功构建重组质粒HTb-dORF5和pNZ8149-dORF5并获得表达,这为进一步研制预防PRRS疫苗奠定坚实基础。The present study was designed to construct recombinant plasmids,which could express porcine reproductive and respiratory syndrome virus（PRRSV）ORF5gene.RNA was extracted from spleen and lung samples of the suspected pigs which were infected with PRRSV.According to PRRSV ORF5 gene,apair of primers was designed for RT-PCR amplification.The ORF5 target gene was cloned into pMD19-T vector and then the recombinant pMD19-ORF5 was achieved.According to the sequencing results and the characteristics of expression vectors,apair of primers with NcoⅠand XbaⅠenzyme cleavage sites was designed.Target fragment dORF5 was amplified and then connected to pProEXHTb and pNZ8149 vectors,respectively.And recombinant HTb-dORF5/DE3 and pNZ8149-dORF5/NZ3900 was induced by IPTG and Nisin,respectively,and analyzed by SDS-PAGE and Western blotting.Recombinant HTb-dORF5/DE3 induced by1.5mmol/L IPTG was expressed in the highest quantity.There were specific band at about 22 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.Recombinant pNZ8149-dORF5/NZ3900 induced by 20ng/mL Nisin was expressed in the highest quantity.There were specific band at about 19 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.The IFA result showed specific green fluorescence.This study successfully constructed recombinant plasmids HTb-dORF5 and pNZ8149-dORF5 and expressed,the result laid a solid foundation for further development of PRRS vaccines.

关 键 词：猪繁殖与呼吸综合征病毒(PRRSV) ORF5基因 表达 大肠杆菌 乳酸乳球菌 

分 类 号：S852.65[农业科学—基础兽医学]