禽腺联病毒VP基因在昆虫细胞中的表达

Expression of VP genes of avian adeno-associated virus in insect cells

为同时在昆虫细胞中按照合适比例表达禽腺联病毒的3个结构蛋白质,首先根据禽腺联病毒VP基因序列设计引物,扩增VP基因,将其克隆至杆状病毒表达载体pFastBac1,获得重组转移载体pFastBac-VP,将其转化到DH10Bac感受态细胞中,经抗性和蓝白斑筛选,获得重组穿梭质粒rBacmid-VP,在脂质体的介导下转染昆虫细胞Sf9,获得重组杆状病毒rBac-VP。SDS-PAGE结果分析表明,3个结构蛋白质VP1、VP2和VP3均获得了表达,分子量正确,且比例为1∶1∶10。Western blot结果显示,表达的重组蛋白质能够与VP3多抗反应。以上结果说明禽腺联病毒的3个结构蛋白质均在昆虫细胞中获得了成功表达。

In order to express three structural proteins of avian adeno-associated virus（AAAV） in an appropriateproportion in insect cells, one pair of specific primers were designed according to the published genome sequences of AAAVto amplify VP genes by PCR, and the amplified fragment was cloned into Baculovirus expression vector pFastBac1. Thenthe recombinant vector pFastBac-VP was transformed into DH10Bac Escherichia. coli, and the positive recombinant bacmidrBacmid-VP was selected after resistance and blue-white plaque screening. rBacmid-VP was transfected into the Sf9 insectcells by liposome, and the rBac-VP was acquired. SDS-PAGE analysis showed three structural proteins, VP1, VP2 and VP3, were expressed in Sf9 with the right molecular weights and the ratio of 1 ：1 ：10. The results of Western blot showedthat the recombinant proteins could be recognized by antisera of VP3.