表达H5N1亚型禽流感病毒HA和NA基因的重组鸭瘟病毒的构建

利用PCR技术扩增出鸭瘟病毒(DPV)生长非必需的TK基因(约1.1kb),将其克隆入pGEM-Teasy载体获得载体pGTK。根据已知的绿色荧光蛋白载体pEGFP-C1的序列设计了一对引物,PCR扩增出pEGFP-C1上含CMV启动子、EGFP及其多克隆位点的完整的基因表达盒插入pGTK的TK上,获得质粒pGTK-EGFP。根据Genbank已发表的H5N1亚型禽流感病毒的血凝素(HA)和神经氨酸酶(NA)基因序列,设计了两对引物,分别从pT-HA和pT-NA两个质粒上扩增出HA和NA基因,克隆到pGTK-EGFP的表达盒的多克隆位点Kpn2I与SmaI之间,构建含EGFP及HA和NA基因的转移质粒载体pGTK-EGFP-HA-NA。将这质粒载体与DPV34F2疫苗毒共转染鸡胚成纤维细胞(CEF),通过荧光方法筛选,获得了表达HA和NA基因的重组DPV(rDPV-HA-NA)。

Construction of recombinant duck plague virus expressing HA and NA gene of H5N1 Subtype avian influenza virus

Using extracted total DNA from duck plague virus (DPV) vaccine strain infected chicken (Gallus domesticus) embryo fibroblasts(CEF) cells as template, the 1.0 kb TK gene nonessential for viral replication was amplified by polymerise chain reaction(PCR) and cloned into T-easy vector to obtain pGTK. According green fluorescent protein vector pEGFP-C1 sequence, one pairs of the primers used to amplify CMV,EGFP and its integrallty gene expression case which lie in the plasmid pEGFP-C1 were inserted into TK site which lies in pGTK,resulting in the transfer vector pGTK-EGFP. Two pairs of primers were synthesized according HA and NA sequence of H5N1 subtype avian influenza virus published on genbank, one pair of the primers used to amplify the HA gene, the other to amplity the NA gene, both were then inserted into the resulting transfer vector pGTK-EGFP in the EGFP multiclonal sites to obtain transfer vector pGTK-EGFP-HA-NA. The recombinant virus,designated rDPV-HA-NA was generated by cotransfection of CEF with pGTK-EGFP-HA-NA and the DPV34F2 vaccine virus.