棉铃虫核型多角体病毒(HaSNPV)G4株Ha109蛋白的原核表达及抗体制备  

Prokaryotic Expression of Ha109 from Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus G4 and Generation of Antibody

作　　者：闫尧[1] 曾利平[1] 钱丽莹[1] 徐旭士[1] 

机构地区：[1]南京师范大学生命科学学院,南京210046

出　　处：《生物技术通报》2011年第5期178-182,共5页Biotechnology Bulletin

摘　　要：根据棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopoly-hedrovirus,HaSNPV)G4株基因组全序列,设计引物,利用PCR方法扩增出开放阅读框(open reading frame,ORF)109编码片段并将其克隆至载体pGEM-TEasy,测序正确后将其亚克隆至原核表达载体pGEX-KG,经IPTG诱导,融合蛋白GST-Ha109在E.coliBL21中以包涵体形式得到高效表达。表达目的蛋白大小为38.5 kD,经SDS-PAGE分离纯化,免疫新西兰大白兔制备了多克隆抗体。抗体经1∶6 000倍稀释后用于Western blotting分析,获得特异性显色信号,为深入研究Ha109的功能奠定基础。The orf109＇s coding region from ATG to UAA was amplified from Helicoverpa armigera single nucleocapsid nucleopoly-hedrovirus genome by PCR.The PCR product was cloned into the pGEM-T Easy vector,then it was subcloned into the prokaryotic expression vector pGEX-KG after sequencing.After IPTG induction,the E.coli BL21 strains containing recombinant plasmids expressed a considerable fusion protein of GST-Ha109 in the form of inclusion body.The molecular weight of the fusion protein was 38.5 kD,which was in agreement with the anticipation.The recombinant protein which was purified by SDS-PAGE was used to immune the New Zealand white rabbit.Western blotting analysis using the multiclonal antibodies with 1∶ 6 000 diluted indicated that these antibodies could react specifically with the GST-Ha109 fusion protein expressed in E.coli BL21.The antibodies were suitable to be used for further function analysis of Ha-109 in infect insect cells in the future.

关 键 词：HASNPV 克隆 多克隆抗体 WESTERN BLOTTING 

分 类 号：S476.13[农业科学—农业昆虫与害虫防治] Q78[农业科学—植物保护]