传染性法氏囊病病毒上海超强毒株VP2-4-3基因真核表达质粒的构建与表达

Construction and Expression of Eukaryotic Expression Plamid of VP 2-4-3 Gene of Very Virulent Infectious Bursal Disease Virus Strain SH95

应用Long and Accurate—PCR(LA-PCR)技术,扩增克隆了鸡传染性法氏囊病病毒上海超强毒(wIB-DV)的VP2—4—3基因。应用粘性末端连接策略,将VP2-4-3基因克隆人真核表达载体pALTER-MAX。经酶切鉴定,表明VP2-4-3基因为正向插入,位于CMV启动子下游。该真核表达质粒在脂质体的介导下转染Vero细胞,经特异性抗IBDV抗体的免疫荧光检测,发现在细胞内有特异蛋白表达。

VP 2-4-3 gene of very virulent infectious bursal disease virus strain SH95 (vvIBDV) was applied by Long and Accurate-PCR(LA-PCR). Then VP2-4-3 gene was ligated into eukaryotic expression vector pALTER-MAX by cohesive ends. It was proved by enzyme-digestion that VP2-4-3 gene has cloned into pALTER-MAX with the right orientation and lies in the downstream of CMW promoter. Vero cells were transfected by the recombinant eukaryotic expression plasmid inducted by Li-pofectamine?000 and specific protein was detected in the cells by immunofluorescent assay of anti-IBDV antibody.