表达新城疫病毒F48E8株血凝素-神经氨酸酶的重组鸡痘病毒的构建和鉴定

CONSTRUCTION AND IDENTIFICATION OF RECOMBINANT FOWLPOX VIRUS FOR EXPRESSING HAEMAGGLUTININNEURAMINDASE GLYCOPROTEIN OF NEWCASTLE DISEASE VIRUS STRAIN F48E8

在克隆和鉴定新城疫病毒（NDV）F48E8株血凝素－神经氨酸酶（HN）基因的基础上，应用分子克隆技术将HN基因导入鸡痘病毒插入载体pFG1175－1中启动子P7．5的下游，得到携带NDV-HN基因的质粒pFGHN1175－1。将此质粒pFGHN1175－1以脂质体转染中国鸡痘病毒疫苗株282E4株感染3～4h的鸡胚成纤维细胞，采用蓝斑筛选方法纯化3次，得到稳定的重组鸡痘病毒。用NDV—HN基因特异性探针进行斑点杂交试验以及用HN基因特异性引物作PCR检测，表明NDV—HN基因已插入鸡痘病毒基因组中。以NDV－HN蛋白特异性单克隆抗体进行间接免疫荧光试验，证实重组鸡痘病毒在感染细胞中表达了HN糖蛋白，从而成功建立了表达新城疫病毒F48E8株血凝素-神经氨酸酶的重组鸡痘病毒。

For the construction of transfer vector prm1175-1, the gene encoding haemaggl uhnin-neuednidas e (HN ) glycoprote in of newc ashedi seaevirus (NDV ) s the n F48E8 was removed from plasndd PGEMHNand inserted int0 the ixndIII site of insenion vector pFG1175-1,downsbeam of P7.5 promotor. Chicken embryo fibroblast (CEF) cell cultUres which had been infected with fowlPOx virus (FPV)chinese vascine strain 282E4 for 3～4hotirs were transfected with pFGHNl 175-1 plasthed DNA by liposomal transfechon. Recombinant FPV with blue plaques were selected and purified 3 hmes in CEF cell cultures overiaid agarose contrining X-gal.PCR analysis and DNA dotblotting hybridization assay indicated that the HN gene had inserted into the FPV genondc DNA. The expression of the NDV HN gene in recombinant rw was confirmed by indirect immtmofluorescence assay with specific monoclonal anhbody.