IBDV多聚蛋白基因真核表达质粒的构建与表达

Construction and Expression of Eukaryotic Expression Plasmid of VP2-4-3 Gene of Very Virulent Infectious Bursal Disease Virus

将IBDV上海超强毒株的多聚蛋白基因(vp2-4-3)克隆入真核表达载体pALTER-MAX,构建成功pALTER-MAX-VP2-4-3真核表达质粒,经纯化后,pALTER-MAX-VP2-4-3在LipofectamieTM2000介导下转染Vero细胞、11日龄鸡胚的绒毛尿囊膜(CAM)和肌肉注射2日龄的雏鸡,1周后,分别提取细胞或组织中的总DNA或总RNA,用DIG标记探针均可检测到阳性杂交信号;转染的Vero细胞飞片和肌肉冰冻切片,进行免疫荧光检测均呈现阳性结果;转染的鸡胚CAM匀浆上清,用兔抗IBDV超强毒的高免血清,经Dot-ELISA检测呈现阳性。表明转染后基因获得表达,表达的蛋白具有免疫反应性。

vp2-4-3 gene of very virulent infectious bursal disease virus strain SH95 (vvIBDV) was double enzyme digested and cloned into eukaryotic expression vector pALTER-MAX by cohesive ends. The recombinant plasmid was identified by enzyme-digestion, which showed that the gese was cloned into pALTER-MAX vector with right orientation and in the downstream of CMV promoter. To express this recombinant eukaryotic expression plasmid, Vero cells were transfected with the plasmids mediated by LipofectamineTM 2000 and specific proteins was detected in the cells by immuno- fluorescent assay of anti-IBDV antibody. 2-day-old chickens were intramuscularly injected with the plasmid pALTER-MAX-VP2-4-3, thigh muscles were collected at 1 week after injection. Protein expression was detected by immuno-fluorescent assay with antibody to IBDV. Expression of plasmid pALTER-MAX-V P2-4-3 in chorioallantoic membrane(CAM)of 11-day-old chicken embryo was detected by Dot-ELISA with antibody to IBDV.