表达H5N1亚型禽流感病毒HA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)生长非必需区的TK基因(约2.5kb),将其克隆入pUC19载体获得载体puDTK。根据已知载体pcDNA-LacZ的序列设计了一对引物,PCR扩增出pcDNA-LacZ上含CMV启动子、多克隆位点、SV40及LacZ的完整的基因表达盒插入puDTK的TK上,获得质粒puDTCL。根据Genbank已发表的H5N1亚型禽流感病毒的血凝素(HA)基因序列,设计引物从T-HA质粒上扩增出HA基因,克隆到puDTCL的表达盒的多克隆位点NheI与ApaI之间,成功构建含LacZ及HA基因的转移载体质粒puDTCL-HA。将这载体质粒与DPV34F2疫苗毒共转染鸡胚成纤维细胞(CEF),经蓝斑克隆筛选和纯化,获得了遗传性状稳定的表达HA基因的重组鸭瘟病毒。

Construction of Recombinant Duck Plague Virus Expressing HA Gene of HSN1 Subtype Avian Influenza Virus

Using extracted total DNA from duck plague virus (DPV) as template,the 2.5kb TK gene nonessential for viral replication were amplified by polymerase chain reaction (PCR) and cloned into pUC19 vecter at one time to obtain puDTK. According the pcDNA-LacZ sequence, one pairs of the primers used to amplify CMV , its integrallty gene case , SV40 and LacZ which lie in the plasmid pcDNA-LacZ were inserted into TK site which lies in puDTK., resulting in the transfer vector pUTCL.One pair of primers was synthesized according HA sequence of H5N1 subtype avian influenza virus published on genebank, which was used to amplify HA gene from T-HA, then was inserted into the resulting transfer vector pUTCL in the multiclonal sites to obtain the pUTCL-HA vector completely. After construction of transfer vector, it was transfected on CEF cells with DPV. Following clonning and purification of blue plaque , the pure HA-fowlpox virus recombinant was obtained.