腺病毒介导O型口蹄疫病毒VP1基因在关中奶山羊乳腺上皮细胞中的表达  

Adenoviral-mediated expression of type O foot-and-mouth disease viral polyprotein gene in mammary epithelial cells of diary goats

作　　者：贺晓丽[1] 林小湲 刘军[1] 王勃 卓伟伟[1] 张涌[1] 郑月茂[1] 

机构地区：[1]西北农林科技大学动物医学院农业部动物生物技术重点开放实验室,陕西杨凌712100 [2]新疆哈密地区动物疫病预防控制中心,新疆哈密839000

出　　处：《中国兽医学报》2016年第9期1537-1543,共7页Chinese Journal of Veterinary Science

基　　金：战略性新兴产业重大产品(群)资助项目(2015KTCQ02-18)

摘　　要：构建O型口蹄疫病毒抗原表位VP1的重组腺病毒载体,并在体外和体内培养试验中检测腺病毒介导VP1的表达。利用PCR技术,从原始质粒中扩增VP1基因片段,双酶切后连接载体构建重组穿梭质粒pHBAd-MCMVVP1-GFP,并经磷酸钙沉淀法携带骨架质粒按比例转染HEK293A细胞,获得P1代腺病毒颗粒,经多轮扩增后进行浓缩纯化,获得复制缺陷型腺病毒颗粒,测定病毒滴度。进行体外感染乳腺上皮细胞,通过GFP的荧光表达量确定最佳感染复数。然后用RT-PCR和Western blotting检测感染的乳腺上皮细胞中抗原表位VP1的表达。进一步用纯化的腺病毒通过乳头滴定法感染羊乳腺组织,检测其乳汁中VP1的表达。结果显示,PCR、双酶切和基因测序等结果表明基因扩增和腺病毒载体构建正确,以其感染关中奶山羊乳腺上皮细胞和乳腺组织时,均可检测到VP1蛋白表达。结果表明,成功构建了含VP1基因的重组腺病毒载体,且能够介导VP1基因在体外和体内培养试验中有效表达,为进一步研究纯化分离的VP1蛋白的抗原性并提高抗原优化技术提供基础研究。To construct a recombinant adenovirus vector containing viral polyprotein VP1 of type O foot-and-mouth disease virus and to explore its expression in vitro and in vivo culture experiments of dairy mammary epithelial cells. VP1 was amplified by PCR from the original plasmid pBT-VP1 and properly integrating into the shuttle pHBAd-MCMV-GFP vector, co-transfecting into HEK293A cells by calcium phosphate transfection method with adenovirus helper plasmid pBH- Glox（delta）E1 to produce pseudovirus. The infective replication defective adenovirus solution was collected by cold-hot repeatedly after the appearance of a severe cytopathic effect and then puri- fied,the determined virus titer was about 1 ×10^13 PFU/mL. The wrapped adenovirus vector was transfected into dairy mammary epithelial cells and tissues. The expression of the VPI gene was detected by using RT-PCR and Western blotting. The expression levels of VP1 protein of FMDV in the milk of goats were detected by the Coomassie blue staining. Constructed adenovirus vector could induce the expression of the VP1 gene in HEK293A cells and mammary epithelial cells.These results would establish the foundation for further study on the recombinant adenovirus alive vector vaccine against serotype O FMD.

关 键 词：VP1 腺病毒表达系统 蛋白表达 乳腺上皮细胞 奶山羊 

分 类 号：S852.65[农业科学—基础兽医学]