棉铃虫核多角体病毒iap3基因表达时相分析

Time Course Analysis of Helicoverpa armigera Nucleopolyhedrovirus iap3 Gene Expression

目的:原核表达棉铃虫核多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HearNPV)iap3基因,制备该蛋白的多克隆抗体,并利用该抗体分析iap3基因在病毒感染过程的表达时相,为深入研究提供基础。方法:PCR扩增iap3基因后,克隆至pET28b,转化到大肠杆菌BL21(DE3)中诱导表达,利用亲合层析进行蛋白纯化,将纯化融合蛋白免疫大鼠制备抗血清,利用抗血清Western blot检测IAP3在病毒感染过程的表达时相。结果:成功在原核细胞中表达iap3基因,并获得纯化的融合His-tag的IAP3蛋白,制备了该蛋白的多克隆抗体。发现iap3基因最早在感染后24h表达,到72h到达表达高峰。结论:获得了IAP3多克隆抗体,iap3基因是一个晚期表达基因。

Objective： To express iap3 gene of Helicoverpa armigera nucleopolyhedrovirus（HearNPV） and to prepare its polyclonal antibody.To analyze the time course expression of iap3 gene in cells infected with HearNPV for further research of iap3 gene.Method： iap3 gene was amplified by PCR.The PCR product was inserted into the expression vector pET-28b.The recombinant vector was transformed into E.coli BL21（DE3） and induced the fusion IAP3 protein expression with IPTG.The fusion IAP3 was purified with affinity chromatography and injected into rat to raise polyclonal antibodies.The time course expression of iap3 gene was analyzed with Western blot using the antibody.Result： The IAP3 was successfully expressed in E.coli and purified as a fusion protein with His-tag.The IAP3 polyclonal antibody was produced from rat.The earliest expression of iap3 gene was detected at 24 hour post-infection.The yield of expression rose to peak at 72 hour post-infection.Conclusion： The polyclonal antibody of IAP3 was prepared.The HearNPV iap3 gene is a late expression gene.