大熊猫犬瘟热病毒H基因重组山羊痘病毒的构建和鉴定  

Construction and Identification of Recombinant Goatpox Virus Expressing CDV H Gene

作　　者：程松[1] 王和兴 郑敏[2] 罗满林[3] 单芬[1] 李婉萍[1] 陈武[1] CHENG Song;WANG Hexing;ZHENG Min;LUO Manhn;SHAN Fen;LI Wanping;CHEN Wu(Guangzhou Zoo, Guangzhou 510070, China;, Guangxi Center for Animal Disease Control and Prevention;Nanning 3. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;Mengzi Animal Disease Prevention and Control Center ,Mengzi 661199, China)

机构地区：[1]广州动物园,广州510070 [2]广西动物疫病预防控制中心,南宁530001 [3]华南农业大学兽医学院,广州510642 [4]红河州农业局红河州动物疫病预防控制中心,蒙自661199

出　　处：《病毒学报》2018年第4期550-556,共7页Chinese Journal of Virology

摘　　要：本研究参照GenBank上已公布的大熊猫犬瘟热病毒（CDV）H编码的基因序列,对其抗原保守结构和识别区域进行修饰,将CDV保护性抗原H基因克隆到pMDTK-pEL载体中,然后运用酶切、连接将表达盒PELH构建到pTK-Eg载体中,从而获得重组转移载体质粒pTK-H-Eg。将重组转移载体质粒pTK-H-Eg与GTPV AV41株共转染BHK细胞,使其在细胞内发生同源重组,获得重组CDV的H基因的山羊痘病毒,然后在Vero细胞上加压筛选,利用gpt培养基筛选到稳定表达EGFP的重组病毒。通过PCR、免疫荧光以及Western Blot鉴定,证明CDV H基因成功构建到GTPV基因组中,并且在细胞中获得了正确表达。本研究获得了表达CDV H基因重组山羊痘病毒,并命名为vpTK-H-Eg。In this study,the H gene sequence of the canine distemper virus（CDV）in giant panda was modified and synthesized based on the published coding sequences on GenBank.First the gene transfer vector pMDTK-PEL was constructed.The H gene from the CDV was cloned into the transfer vector pMDTKPEL through digestion of restriction enzyme,H Gene Expression Cassettes were inserted into the vector pTK-Eg,and we obtained the recombinant transfer vector plasmid pTK-H-Eg.The recombinant transfer vector plasmid was transfected into BHK cells that had been infected with the goatpox virus,through homologous recombination recombinant goatpox virus was generated.The recombinant goatpox virus containing the H gene of the goatpox virus cultured in Vero cells underwent pressure selection by adding mycophenolic acid to the culture.After purification and polymerase chain reaction,the immunofluorescence test and Western Blot demonstrated that the H gene of the CDV had been inserted into the goatpox virus and correctly expressed the H protein of the CDV in cells.We obtained the recombinant GTPV AV41 and name it＂vpTK-H-Eg＂.

关 键 词：犬瘟热病毒(CDV) 同源重组 重组山羊痘病毒 

分 类 号：S852.65[农业科学—基础兽医学]