Cloning,Expression of Autographa californica nucleopolyhedro virus vp39 Gene in Escherichia coli and Preparation of Its Antibody
Cloning,Expression of Autographa californica nucleopolyhedro virus vp39 Gene in Escherichia coli and Preparation of Its Antibody
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发表时间：
2007
期刊：
影响因子：
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通讯作者：
P. Yi
中科院分区：
文献类型：
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作者：
P. Yi
目的：构建苜蓿银纹夜蛾核多角体病毒（AcMNPV）VP39原核表达质粒并表达和纯化蛋白，用于制备多克隆抗体。方法：采用PCR法扩增vp39基因。将PCR产物克隆至表达载体pET-21a(+)中，并转化大肠杆菌BL21(DE3)。 SDS-PAGE分离融合蛋白，凝胶提取回收。纯化蛋白免疫大白兔，Western blot检测抗体。结果：构建表达质粒pET-vp39。 BL21(DE3)菌株含有vp39重组质粒，经IPTG(异丙硫基-β-D-半乳糖苷)诱导后，表达40kDa T7·Tag融合蛋白。 Western blotting分析表明该抗体能够与AcMNPV感染的Sf9细胞中存在的相应蛋白发生反应。结论：本研究制备了抗AcMNPV-VP39多克隆抗体，可作为进一步研究病毒感染过程中VP39与宿主因子相互作用的检测工具。
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.