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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 prokaryotic express the iap3 gene of Helicoverpa armigera nucleopolyhedrovirus (HearNPV), and to prepare the polyclonal antibody of this protein. The expression of iap3 gene during virus infection can be analyzed by this antibody and provide the basis for further study. Methods: After iap3 gene was amplified by PCR, it was cloned into pET28b and transformed into E. coli BL21 (DE3) to induce expression. The protein was purified by affinity chromatography. The purified fusion protein was immunized to prepare antiserum. The phase of expression of IAP3 during virus infection was examined. Results: The iap3 gene was successfully expressed in prokaryotic cells and the purified His-tag fused IAP3 protein was obtained. The polyclonal antibody of this protein was prepared. The iap3 gene was found to be expressed at the earliest 24 hours after infection and peaked at 72 hours. Conclusion: IAP3 polyclonal antibody was obtained, iap3 gene is a late expression gene.