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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
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)
关键词
小反刍兽疫病毒
山羊痘病毒
H基因
F基因
peste des petits ruminants virus(PPRV)
goatpox virus(GPV)
H gene
F gene
