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程松1 王和兴2 郑敏3 罗满林4 单芬1 李婉萍1 陈武1

(1.广州动物园, 广州 510070)
(2.红河州农业局红河州动物疫病预防控制中心, 蒙自 661199)
(3.广西动物疫病预防控制中心, 南宁 530001)
(4.华南农业大学兽医学院, 广州 510642)
【创新点】通过同源重组,10轮加压筛选纯化、PCR与Western Blot的鉴定,成功获得了表达大熊猫犬瘟热病毒H基因重组病毒vpTK-H-Eg。

【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。

【关键词】 犬瘟热病毒(CDV);同源重组;重组山羊痘病毒;


Construction and Identification of Recombinant Goatpox Virus Expressing CDV H Gene

CHENG Song1 WANG Hexing2 ZHENG Min3 LUO Manlin4 SHAN Fen1 LI Wanping1 CHEN Wu1

(1.Guangzhou Zoo, Guangzhou, China 510070)
(2.Mengzi Animal Disease Prevention and Control Center, Mengzi, China 661199)
(3.Guangxi Center for Animal Disease Control and Prevention, Nanning, China 530001)
(4.College of Veterinary Medicine, South China Agricultural University, Guangzhou, China 510642)
【Novelty】Through homologous recombination recombinant goatpox virus was generated after purification and polymerase chain reaction. The immunofluorescence test and western blotting demonstrated that the H gene of the Canine distemper virus(CDV) had been inserted into the goatpox virus and correctly expressed the H protein of the CDV in cells.The recombinant GTPV AV41 is named as vpTK-H-Eg.

【Abstract】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 pMDTK-PEL through digestion with restriction enzymes, then the H gene expression cassettes were inserted into the vector pTK-Eg, thus we obtained the recombinant transfer vector pTK-H-Eg. The recombinant transfer vector was transfected into BHK cells that had been infected with the goatpox virus. The recombinant goatpox virus containing the H gene of CDV was cultured in Vero cells which then underwent pressure selection by adding mycophenolic acid to the culture. After purification and polymerase chain reaction, the immunofluorescence test and Western blotting demonstrated that the H gene of CDV had been inserted into the goatpox virus and correctly expressed the H protein in cells. We obtained the recombinant GTPV AV41 and name it vpTK-H-Eg.

【Keywords】 Canine distemper virus (CDV); Homologous recombination; Recombined goatpox virus;


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This Article


CN: 11-1865/R

Vol 34, No. 04, Pages 550-556

June 2018


Article Outline



  • Materials and methods
  • Results
  • Discussion
  • References