利用杆状病毒Bac-to-Bac系统在家蚕表达猪繁殖与呼吸综合征病毒的E蛋白和M蛋白

猪繁殖与呼吸综合征病毒(PRRSV)是一种严重危害养猪业的传染性病原。PRRSV病毒的E蛋白和M蛋白基因是开发PRRSV病毒新型疫苗的目标基因。利用杆状病毒Bac-to-Bac表达系统,将PRRSV病毒的E蛋白和M蛋白的基因亚克隆到杆状病毒转移载体pFastBacHTb中,获得重组转移质粒pFastBacHTb-E和pFastBacHTb-M,转化大肠杆菌Bm DH10 Bac感受态细胞,获得重组杆粒BmNPV Bacmid-E、BmNPV Bacmid-M,将这些重组杆粒转染家蚕培养细胞Bm5,获得重组病毒BmNPV-E和BmNPV-M。将2种重组病毒分别接种5龄起蚕,用SDS-PAGE和Western blotting方法在重组Bacmid DNA转染的Bm5细胞和感染重组病毒的家蚕幼虫血细胞中分别检测到分子质量约20 kD和18 kD的E蛋白和M蛋白,表明PRRSV病毒的E蛋白和M蛋白在家蚕培养细胞及幼虫体内获得了表达,为利用家蚕-杆状病毒表达系统研制PRRSV的新型疫苗与诊断试剂奠定了基础。

Expression of E Protein and M Protein of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)in Bombyx mori Using Baculovirus Bac-to-Bac System

Porcine reproductive and respiratory syndrome virus(PRRSV) is a kind of infectious pathogen that greatly en-dangers swine industry.E protein and M protein genes of PRRSV are target genes for developing novel vaccines against PRRSV.By using baculovisus Bac-to-Bac expression system,E protein and M protein genes of PRRSV were subcloned into a baculovirus transfer vector pFastBacHTb to yield the recombinant transfer plasmids pFastBacHTb-E and pFast-BacHTb-M.Recombinant bacmids BmNPV Bacmid-E and BmNPV Bacmid-M were obtained after transforming the E.coli Bm DH10 Bac competent cells.Recombinant viruses BmNPV-E and BmNPV-M were obtained after the bacmids were used to transfect cultured Bombyx mori cell line Bm5.After the two recombinant viruses were used to inoculate newly exu-viated Bombyx mori larvae of the 5th instar,SDS-PAGE and Western blotting detected E and M proteins with molecular weight of approximately 20 kD and 18 kD respectively in Bm5 cells transfected by recombinant Bacmid DNA and in hemocytes of Bombyx mori larvae infected by the recombinant virus,indicating that E protein and M protein had been successfully expressed inside Bombyx mori cells and larvae.The above results establish a good basis for developing novel vaccines and diagnostic agents against PRRSV by using Bombyx mori-baculovirus expression system.