H9亚型禽流感病毒血凝素基因重组禽痘病毒的构建

Construction of recombinant fowlpox virus expressing haemagglutinin gene of H_9 avian influenza virus

【目的】获得表达H9亚型禽流感病毒血凝素(HA)基因的重组禽痘病毒。【方法】将含禽痘病毒启动子LP2EP2驱动的HA基因,插入到禽痘病毒转移载体pSY681中,获得重组转移载体pSY681/HA。用脂质体将其转染已感染亲本禽痘病毒S-FPV-017株的鸡胚成纤维细胞,使其在鸡胚成纤维细胞内与禽痘病毒基因组发生同源重组,产生表达HA的重组禽痘病毒rFPV-HA。在含有X-gal的营养琼脂培养基上进行蓝斑筛选后,对重组病毒进行多次蚀斑克隆,并用间接免疫荧光法对感染重组病毒鸡胚成纤维细胞中HA的表达产物进行鉴定。【结果】以重组禽痘病毒DNA为模板,利用HA基因特异引物进行PCR,扩增出1条约1.7 kb左右的带。以间接免疫荧光法证实重组禽痘病毒能表达HA。【结论】成功构建了表达H9亚型禽流感病毒HA基因的重组禽痘病毒,且构建的重组禽痘病毒能表达具有生物学活性的HA。

[Objective] Recombinant fowlpox viruses （rFPV） were developed expressing H9 avian influenza virus （AIV） haemagglutinin （HA）. [Method] HA cDNA was cloned into BamH Ⅰ site of pSY538 plasmid and then subcloned into Sfi Ⅰ site of pSY681 plasmid containing LacZ gene. The plasmid,pSY681/ HA,was used to transfect on the chicken embryo fibroblasts cell （CEF） pre-infected with fowlpox virus S- FPV-017. By selection of blue plaques on the CEF overlaid with agar containing X-gal, the rFPV-HA recombinants were obtained and identified by restriction enzyme analysis and PCR. HA in the recombinalt vi- rus-infected CEF was indentified by indirect immune of luorescence assay. [Result] The results indicated that recombinant fowlpox viruses contained HA gene. The expression of HA was detected in the recombinant virus-infected CEF by indirect immunofluorescence using antibody against AIV. [Conclusion] The recombinant fowlpox virus （rFPV-HA） was developed and had biological activity.