【摘要】 目的:构建苜蓿丫纹夜蛾核多角体病毒(Autographa californica nucleopolyhedro virus,AcMNPV)VP39的原核表达载体,表达、纯化蛋白并制备多克隆抗体。方法:用PCR方法扩增vp39基因,并将其克隆至pET-21a(+)上,转化到大肠杆菌BL21(DE3)中进行诱导表达,采用割胶回收的方法纯化融合蛋白,纯化的融合蛋白作为抗原,免疫新西兰大白兔,Western blot检测抗体活性。结果:构建了pET-VP39原核表达质粒,含有该质粒的大肠杆菌经IPTG诱导超量表达了一个与预期理论值相符的约为40kDa的融合蛋白。对制备的抗体进行免疫印迹分析表明该抗血清能与感染苜蓿丫纹夜蛾核多角体病毒的细胞蛋白样品发生特异性反应。结论:获得了兔抗AcMNPV-VP39多克隆抗体,为进一步深入研究VP39在病毒侵染过程中与宿主因子的相互作用提供了检测工具。更多还原

【Abstract】 Objective:To construct Autographa californica nucleopolyhedro virus (AcMNPV) VP39 prokaryotic expression plasmid and express and purify protein for preparation of polyclonal antibody. Methods: vp39 gene was amplified by PCR. The PCR product was cloned into the expression vector pET-21a(+) and transformed into Escherichia coli BL21(DE3). The fusion protein was separated on SDS-PAGE and recovered by gel extraction.White rabbits were immunized with the purified protein.The antibody was detected by Western blot. Results:The expression plasmid pET-vp39 was constructed. The BL21(DE3) strain, containing vp39 recombinant plasmid, expressed a 40kDa T7·Tag fusion protein after induction with IPTG (isopropylthio-β-D-galactoside). Western blotting analysis indicated that the antibody could react with the corresponding protein existed in AcMNPV-infected Sf9 cells. Conclusion:In this study, polyclonal anti-AcMNPV-VP39 antibody has been prepared, which can be used as the detection tool for further research of the interaction of VP39 with host factors during viral infection.更多还原