表达H5亚型禽流感病毒HA基因的重组马立克氏病病毒的构建

采用聚合酶链反应或反转录聚合酶链反应扩增出H5亚型禽流感病毒(AIV)的HA基因、网状内皮增生症病毒的长末端重复序列(LTR)、马立克氏病病毒(MDV)Rispens CVI988毒株基因组的sorf 1和sorf 2序列、两端带loxp位点的lac/smGFP标志基因,构建含这些基因的转移载体质粒pMHA;以MDV Rispens CVI988毒株的基因组DNA和PMHA质粒DNA共转染鸡胚成纤维细胞(CEF),采用同源重组方法将LTR、lac/smGFP和HA基因插入到MDV基因组,获得重组病毒rMDV-HA/GFP;以cre介导的同源重组去除lac/smGFP标志基因,再转染CEF,获得仅带LTR启动子和HA基因的重组MDV疫苗毒株rMDV-HA.rMDV-HA仍保留了MDV RispensCVI988疫苗毒株的复制特点,并能稳定表达AIV的HA.

Construction of a recombinant Marek's disease virus expressing HA gene of H5 AIV

The transfer plasmid for generating rMDVs,pMHA,was constructed containing lac/smGFP flanked by loxP and HA gene of highly pathogenic AIV of H5 subtype under the control of the reticuloendotheliosis virus(REV) long terminal repeat(LTR),sorf1-sorf2 gene of MDV Rispens CVI988.The transfer plasmid pMHA was tranfected the chicken embryo fibroblast(CEF) cells infected with MDV Rispens CVI988 strain to generate the recombinant virus rMDV-HA/GFP,which inserted the sequence of AIV HA and Lac/smGFP genes into the MDV genome by homogeneous recombination.The Lac/smGFP gene was deleted from rMDV-HA/GFP by cre-based homogeneous recombination to generate the recombinant virus rMDV-HA,which contains HA gene under the control of REV LTR.rMDV-HA remains the similar replication characteristic of MDV Rispens CVI988 strain and is stable for the expression of HA protein of AIV in vivo.