猪繁殖与呼吸综合征病毒GP5和M基因重组腺病毒的构建及表达  

Construction and Expression of the Recombinant Adenovirus GP5 and M Protein of PRRSV

作　　者：高博[1,2] 马玲[1] 张念[1,3] 毛燕[1] 陈凤莲[1] 张守峰[2] 刘烨[2] 张菲[2] 扈荣良[2] 吴健敏[1] 

机构地区：[1]广西兽医研究所广西畜禽疫苗重点实验室,广西南宁530001 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《动物医学进展》2012年第1期23-27,共5页Progress In Veterinary Medicine

基　　金：广西青年科学基金(桂科青0991064);广西自然科学基金(桂科自0991219);广西科学研究与技术开发计划(桂科攻0815009-3-5)

摘　　要：为构建猪繁殖与呼吸综合征病毒(PRRSV)重组腺病毒活载体疫苗,本研究将PRRSV GP5和M基因串联表达盒定向克隆至穿梭质粒pacAd5CMVK-NpA多克隆位点处,获得重组穿梭质粒pacAd5CMVK-NpA-GP5-M,与骨架质粒pacAd5 9.2-100共转染293AD细胞,包装出含有GP5和M基因的重组腺病毒rAd5-GP5-M。应用PCR技术、间接免疫荧光、Western blot分析,结果表明,GP5和M基因已经被成功整合到了腺病毒基因组上,且GP5和M蛋白在293AD细胞上均得到了表达;滴度测定表明,该重组腺病毒rAd5v-GP5-M的TCID50为10-6.5/mL;连续传代试验表明该重组腺病毒可在细胞内稳定繁殖,有效地转录并表达GP5和M蛋白,具有较高的遗传稳定性。该研究为PRRSV重组腺病毒疫苗的研制打下了基础。In order to construct the recombinant vaccine of Porcine reproductive and respiratory syndrome virus（PRRSV）,in this research,the ORF which contained GP5 and M genes was cloned into the shuttle vector pacAd5CMVK-NpA named pacAd5 CMVK-NpA-GP5-M and contransinfeced into 293AD cells with the backbone vector pacAd5 9.2-100.The recombinant adenovirus rAd5v-GP5-M was packed after the homologous recombination in the cells.Subsequently the recombinant virus was detected by PCR,indirect immunofluorescence test and Western blot assay.The results indicated the GP5 and M gene were intergrated into the adenovirus geneome and the proteins GP5 and M all were expressed in 293AD cells.The titre determination tests indicated the TCID50 is 10-6.5/mL.This study established the fundation for the development of recombinant adenovirus vaccine against PRRS.

关 键 词：猪繁殖与呼吸综合征病毒 GP5基因 M基因 重组腺病毒 

分 类 号：S852.659.6[农业科学—基础兽医学] Q789[农业科学—兽医学]