高致病性血清4型禽腺病毒hexon基因在昆虫细胞中的表达及鉴定

Expression and identification of high pathogenic fowl adenovirus serotype 4 hexon gene in insect cell

为获得杆状病毒表达的重组高致病性血清4型禽腺病毒(Fowl adenovirus serotype 4,FAd V-4)六邻体蛋白(Hexon),本研究通过PCR从高致病性血清4型禽腺病毒基因组中扩增到hexon基因,克隆到杆状病毒转移载体p Fast Bac-1,获得重组转移质粒p Fast Bac-hexon,将其转化DH10Bac大肠杆菌并筛选,获得重组杆状病毒穿梭质粒r Bacmid-hexon,将重组杆状病毒穿梭质粒转染Sf9细胞,获得重组杆状病毒r BV-hexon。间接免疫荧光试验和免疫印迹试验结果显示,Sf9细胞中的Hexon蛋白能够与血清4型禽腺病毒阳性血清发生特异性反应,蛋白质分子量约110 000,表明Hexon蛋白在Sf9细胞中获得良好的表达。

To abtain the expression product of high pathogenic fowl adenovirus serotype 4（ FAd V-4） hexon protein,hexon gene was amplified from genome of high pathogenic fowl adenovirus serotype 4,and it was cloned into transfer vector p Fast Bac-1. The recombinant transfer plasmid p Fast Bac-hexon was generated,transformed into DH10 Bac Escherichia coli and screened to obtain recombinant shuttle plasmid r Bacmid-hexon. The r Bacmid-hexon was transfected into Sf9 cell to produce recombinant virus r BV-hexon. The expression of hexon was identified by indirect immunofluorescence assay and western blot,the results showed the recombinant virus in Sf9 cell can be recognized specifically by polyclonal antibody against FAd V-4,and the molecular weight was about 110 000,these results indicated that the hexon protein was well expressed in Sf9 cell.