表达新城疫病毒融合蛋白基因禽痘病毒重组体的构建

通过RT-PCR扩增新城疫病毒F基因,利用禽痘病毒复合启动子、SV40 PolyA终止信号序列构建F基因表达盒。将F基因表达盒与loxp-GFP-loxP序列串联插入禽痘病毒重组臂,构建了禽痘病毒转移质粒载体。在脂质体介导下转染CEF,获得了带有绿色荧光信号的重组病毒rFPVFGFP。通过二次转染,利用Cre-loxP位点特异性重组将重组病毒基因组的GFP自动去除,结果获得了只含新城疫病毒F基因表达盒的重组禽痘病毒rFPVF。试验结果表明,rFPVF复制稳定,表达的F蛋白具有免疫原性,能够刺激鸡只产生抗新城疫病毒的中和抗体。

Construction of recombinant fowlpox virus expressing fusion gene of Newcastle disease virus

The gene expression cassette was constructed with the fusion protein gene fragment from Newcastle disease virus(NDV) by RT-PCR,along with compound promoters of fowlpox virus and SV40 polyA tail signal sequence.In the interaction of liposome,the recombinant virus rFPVFGFP was generated via co-transfection of the fowlpox virus genome and the delivery vector,in which tandem insertion of the fusion gene cassette and loxp-GFP-loxP sequence into the homologous arm gene of fowlpox virus,into chick embryo fibroblast cell.After second transfection,the GFP gene in recombinant genome was removed automatically by site specific recombination of Cre-loxP system and then the rFPVF was gained only containing fusion gene of NDV.The rFPVF recombinant could multiplicate stably in CEF and the fusion protein expression product got immunogenic ability to promote the chicken to produce neutralizing antibody against NDV.