杆状病毒基因Ac108的克隆和原核表达

Cloning and Expression of Baculovirus Ac108 Gene in Escherichia coli

目的:克隆和表达苜蓿丫纹夜蛾核多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus,AcMNPV)开放阅读框108(open reading frame,ORF108,Ac108)基因,为进一步研究其功能打下基础。方法:将Ac108基因克隆至原核表达载体pQE30,在大肠杆菌M15中表达AC108蛋白。以纯化的AC108蛋白作为抗原,免疫新西兰大白兔制备蛋白的多克隆抗体。用该抗体检测了该蛋白在病毒感染细胞中的表达。结果:实现了AC108蛋白的表达,获得了AC108蛋白的多克隆抗体,Western blot检测发现在AcMNPV感染的Sf-9细胞中有一条大小约为27kDa的杂交带,远大于预期的11kDa。结论:AC108蛋白可能以同源或异源聚合物形式在电场中迁移。

Objective： To clone and express Ac108 gene, open reading frame 108（ORF108, Ac108）of Autographa californica muhicapsid nudeopolyhedrovirus （AcMNPV）, which laid the foundation of furthcr function analysis. Methods： Ac108 gene was obtained by PCR from AcMNPV geneme and cloned into prokaryotic expression vector pQE30, then expressed constantly in Escherichia coli M15. Purified AC108 recombinant protein was used as the immunogen to raise polyclonal serum in the New Zealand rabbit and extracts from AcMNPV- infected Sf- 9 cells were analysed by Western blot. Results： AC108 was expressed successfully in M15 and its polyclonal antibody was obtained from the rabbit, a specific polypeptide with an apparent size of 27 kDa was revealed in AcMNPV - infected Sf- 9 cells by westem blot when using the antiserum, which was larger than the predicted size 11 kDa of the putative Acl08 translation product. Conclusion： AC108 protein eleetrophoresed as a homo- or heterooligonaeric complex.