表达鸡新城疫病毒F蛋白的重组马立克氏病毒的构建及其鉴定

Construction and identification of recombinant Marek's disease virus vaccine strain 814 expressing the F protein of NDV

为构建表达鸡新城疫病毒(NDV)融合蛋白(F)的重组马立克氏病毒(MDV),本研究采用RT-PCR方法从NDV强毒株F48E9基因组中扩增出病毒的融合蛋白F基因,构建由CMV启动子和BGH polyA组成的2.7 kb F基因表达盒。将其插入带有黄嘌呤-鸟嘌呤磷酸核糖基转移酶基因(gpt)和MDV US2同源臂的中间转移载体pUAB-gpt中获得重组MDV转移载体pUAB-gpt-wF。将该转移载体与MDV-814疫苗株感染的鸡胚成纤维细胞(CEF)总DNA共转染CEF,经同源重组及gpt选择系统筛选,获得带有F基因表达盒的重组MDV(rMDV814-wF)。其体外增殖与亲本病毒没有差异。经间接免疫荧光试验、PCR、Southern-blot及western blot等试验证明,重组病毒在CEF中传至13代以上仍稳定表达NDV的F蛋白。该重组病毒的构建为MDV活载体疫苗的筛选及应用奠定了基础。

To generate the recombinant Marek＇s disease virus （MDV） expressing F protein of Newcastle disease virus （NDV）, the F gene was amplified by RT-PCR from NDV F48E9 strain. The cassette containing the F gene under the control of the CMV promoter and BGH ployA was constructed and inserted into plasmid pUAB-gpt containing gpt and MDV US2 homologous arms to construct transfer vector of pUAB-gpt-wF. The recombinant MDV was generated by co-transfection chicken embryo fibroblast （CEF） with pUAB-gpt-wF and the total DNA extracted from CEF infected with MDV-814 strain, and selected in present of mycophenolic acid substrates for HGPRT transferase. The growth rate of the recombinant MDV was similar to the parental virus in vitro. Immunefluorenscence assays, western blot and southern blot analysis indicated the recombinant virus was stably expressed NDV F protein over 13 passages in CEF.