共表达新城疫病毒F基因和H9亚型禽流感病毒HA基因的重组鸡痘病毒

应用RT PCR扩增出新城疫病毒F4 8E8株融合蛋白 (F)基因 ,将其克隆入pGEM Teasyvector构建重组质粒pGEM TF并进行测序确证。分别从pGEM T和pUCHA切下F基因和H9亚型禽流感病毒F株 (A chicken china F 1 998)血凝素 (HA)基因 ,通过一系列分子生物学操作步骤插入到质粒pFPV7S中的鸡痘病毒基因组复制非必需片段构建重组质粒p7SHF ,其中F基因和HA基因分别由鸡痘病毒启动子PE L和合成启动子PS调控。最后将P1 1 LacZ报告基因表达盒插入质粒p7SHF获得转移载体pFPVHF ,用以转染已预先感染鸡痘病毒 2 82E4疫苗株的鸡胚成纤维细胞 (CEF)。通过在含有X Gal的营养琼脂上连续挑选蓝色病毒蚀斑获得并纯化重组病毒。PCR和Southernblot检测证实了F基因和HA基因已插入鸡痘病毒的基因组 ;间接免疫荧光试验结果表明重组病毒能够同时正确表达HA和F蛋白。

Recombinant Fowlpox Virus Expressing F of Newcastle Disease Virus and HA of Subtype H9N2 of Avian Influenza Virus

The fusion protein (F) gene of Newcastle disease virus strain F48E8 was obtained by PCR and its sequence was determined. To form recombinant plasmid p7SHF, the F gene in pGEM-TF and the hemagglutinin (HA) gene of AIV, A/Chicken/China/F/1998(H9N2) in pUCHA were cut and inserted into the randomly selected nonessential regions of the fowlpox virus (FPV) under the control of fowlpox virus promoter P E/L and the synthetic promoter Ps respectively. Then the P11-LacZ reporter gene from pppG18 was cloned into p7SHF resulting in recombinant transferring vector pFPVHF, which was transfected into chicken embryo fibroblast (CEF) monolayers pre-infected with Chinese vaccine strain 282E4 of FPV to generate recombinant fowlpox virus co-expressing HA and F (rFPV-HA-F). By selection of blue plaques on the CEF overlaid with agar containing X-gal, rFPV-HF-F was screened and purified. PCR and southern blot assays indicated that the F and HA genes had been inserted into the genomic DNA of FPV. Subsequently expression of F and HA in CEF infected with rFPV-HA-F was confirmed by indirect immunofluorescence assay.