小反刍兽疫病毒H基因或F基因重组山羊痘病毒活载体疫苗的研究  

Study on recombinant goatpox virus vaccines vectored with H or F gene of peste des petits ruminants virus

作　　者：李继东[1,2] 蒙学莲[1] 朱学亮[1] 李雪强[1] 尤亚南 窦永喜[1] 骆学农[1] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部兽医公共卫生重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [2]宁夏大学农学院,宁夏银川750021

出　　处：《中国兽医科学》2015年第2期111-117,共7页Chinese Veterinary Science

基　　金：国家高技术研究发展计划(863)项目(2011AA10A211-1);现代农业产业技术体系建设专项(CARS-40-10)

摘　　要：为构建能够表达小反刍兽疫病毒(PPRV)H基因或F基因的重组山羊痘病毒(GPV)并以此为活载体疫苗,在通用转移质粒pTKfpgigp的多克隆位点中插入PPRV的H基因或F基因,构建重组转移质粒pTKfpgigp-PPRV H和pTKfpgigp-PPRV F,并用Lipofectamine 2000转染已感染GPV的原代绵羊睾丸细胞,经同源重组产生重组GPV(rGPV)。用GFP和gpt选择标记筛选纯化rGPV,通过RT-PCR和免疫荧光技术检测rGPV在原代绵羊睾丸细胞中外源基因的表达情况。将获得的rGPV皮内接种山羊,检测抗山羊痘病毒和小反刍兽疫病毒的特异性抗体的变化情况。结果显示,获得了含有PPRV H基因或F基因的病毒株rGPV-PPRV H和rGPV-PPRV F;在原代绵羊睾丸细胞中rGPV所含的外源基因进行了正常转录和翻译,表达产物能与PPRV抗血清产生特异性反应,动物免疫试验表明,2株rGPV能诱导产生高水平的抗山羊痘病毒和小反刍兽疫病毒的特异性抗体。上述研究结果为PPRV重组标记疫苗的研制提供了参考。In order to construct recombinant goatpox viruses vectored with peste des petits ruminants virus(PPRV)H or F gene and to evaluate their immune effectiveness as vaccine candidates,the constructed plasmids pTKfpgigp-PPRV H and pTKfpgigp-PPRV F containing PPRV H or F gene were transfected into lamb testis cells and infected with GPV previously using Lipofectamine 2000 respectively.The rGPVs were screened by GFP and gpt markers.The expression of H or F gene in the rGPVs was detected by RT-PCR and immunofluorescence.Finally,the rGPVs were vaccinated into goats through intradermal injection and the antibody specificity was analyzed by the anti-GPV or anti-PPRV antibodies.In result,two rGPV-PPRV H and rGPV-PPRV F lines were obtained,and two rGPVs containing the H or F protein were expressed in lamb testis cells.Furthermore,the expressed product showed highly specific reaction with anti GPV and PPRV antibodies,respectively.The above-mentioned result provided a reference for the development of recombinant marker vaccines against PPRV.

关 键 词：小反刍兽疫病毒 山羊痘病毒 H基因 F基因 

分 类 号：S852.65[农业科学—基础兽医学]