利用Cre-loxP自动敲除H5亚型禽流感病毒血凝素重组鸡痘病毒中的报告基因

研究去除重组鸡痘病毒中的报告基因,构建一株只含目的基因的重组毒。将H5亚型AIV的HA基因作为靶基因,两侧含loxP序列的GFP表达盒插入鸡痘病毒重组臂基因构建了转移质粒载体,将其与脂质体混合转染CEF细胞,获得了表达H5和GFP的鸡痘病毒重组体。通过二次转染,利用Cre酶自动敲除重组病毒中的GFP基因,最终获得了只含H5血凝素基因表达盒的重组鸡痘病毒。免疫荧光和病毒滴度测定结果表明,经过连续传代后重组病毒仍然稳定复制并表达H5血凝素。用10⁵PFU和2×10⁵PFU rFPV H5免疫SPF鸡,28d后,免疫组鸡抗体平均滴度(HI)分别达到4log 2和4.5log 2,结果表明,H5HA基因重组病毒能刺激鸡群产生较高特异抗体。

Self-excising reporter gene in recombinant Fowlpox virus expressing H5 hemagglutinin gene of influenza A virus using Cre-loxp systerm

Hemagglutinin gene of subtype H5 avian influenza virus was amplified by polymerase chain reaction to construct expression cassette containing FPV early, late promoter and SV40 polyA tail. Then delivery vector was constructed by subcloning hemagglutinin gene of subtype H5 and GFP gene into fowlpox virus recombinant arm. The delivery vector and Lipid were transfected into CEF cells preinfected with FPV 282E4 strain virus. Recombinant fowlpox virus expressing the green fluorescence protein and hemagglutinin gene was screened and plaques were purified in CEF cell. After a second cotransfection with Cre recombinase plasmid, a recombinant virus only including hemagglutinin gene was gained. The immunofluorescent assay and replication efficiency of virus proved the recombinant could replicate steadily and express subtype H5 hemagglutinin gene. Two groups of 8-day-old SPF chickens were vaccinated with rFPVH5 by the wing-web method at the dosage of 10(5) PFU and 2 x 10(5) PFU respectively. After 28 days,antibodies titer was tested by HI. The results showed that the recombinant fowlpox virus could activate high antibody response.