杆状病毒ac68基因的克隆、表达与抗体制备

Cloning and Expression of Autographa californica Multicapsid Nucleopolyhedrovirus ORF68 Gene in Escherichia coli and Preparation of Its Antibody

目的:对苜蓿丫纹夜蛾核多角体病毒(Autographa califorica multicapsid nucleopoly hedrovirus,AcMNPV)开放阅读框68(open reading frame,ORF68,ac68)进行原核表达,制备该蛋白的多克隆抗体,为深入研究其功能提供基础。方法:将ac68基因克隆至原核表达载体pET28a上,在大肠杆菌BL21(DE3)中表达Ac68蛋白,通过His抗体检测进一步验证所表达的蛋白为带有组氨酸的融合蛋白。以纯化的Ac68蛋白作为抗原,免疫昆明小鼠制备多克隆抗体。结果:实现了ac68基因的原核表达,获得了该蛋白的多克隆抗体并在AcMNPV感染的Sf-9细胞中检测到一条大小为25kD左右的特异杂交带。结论:获得的抗体可用于Ac68蛋白功能的进一步研究。

Objective：To express ac68 gene,open reading frame 68（ORF68,ac68） of Autographa californica nucleopolyhedrovirus（AcMNPV） and to prepare its antibody for further function analysis of ac68 gene.Method：Ac68 gene was amplified from AcMNPV genome by PCR.PCR product was cloned into the expression vector pET28a and transformed into E.coli BL21（DE3）.Ac68 was expressed with the induction of IPTG and the analysis of western blot against 6 × His tag confirmed the expression of Ac68 fused with 6 × His tag.The fusion protein was injected into Kunming mice to raise polyclonal antibodies.Result：Ac68 was expressed successfully in E.coli and its polyclonal antibody was harvested from rats.Conclusion：Ac68-specific antibody can be used for Ac68 function study.