畜牧兽医学报 ›› 2010, Vol. 41 ›› Issue (4): 434-441.doi:

• 预防兽医 • 上一篇    下一篇

表达O型口蹄疫病毒P12A3C基因的山羊痘病毒弱毒株的构建及筛选

王建科,张云德,张强*,吴国华,颜新敏,朱海霞,李健,邵长春,朱彩珠,吴磊
  

  1. 中国农业科学院兰州兽医研究所 家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室,兰州 730046
  • 收稿日期:2009-02-26 修回日期:1900-01-01 出版日期:2010-04-25 发布日期:2010-04-25
  • 通讯作者: 张强

Construction and Screening of the Recombinant Attenuated CPV Expressing P12A3C Gene of FMDV Serotype O

WANG Jianke, ZHANG Yunde, ZHANG Qiang*, WU Guohua, YAN Xinmin,ZHU Haixia, LI Jian, SHAO Changchun, ZHU Caizhu, WU Lei
  

  1. Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
  • Received:2009-02-26 Revised:1900-01-01 Online:2010-04-25 Published:2010-04-25
  • Contact: ZHANG Qiang

摘要: 为构建和筛选表达O型口蹄疫病毒P12A3C基因的山羊痘病毒弱毒株,用已构建的口蹄疫病毒 O/China99 毒株的 EGFPP7.5P12A3C 基因整体通过平末端连接到 KpnⅠ酶切后的线性载体 pUC119TK 中,得到重组载体 pUC119TKEGFPP7.5P12A3C。重组载体通过缺失的 TK 基因与羊痘病毒弱毒株在 BHK21 细胞中同源重组,用 EGFP作为标记筛选出重组毒株,并进行PCR 鉴定、抗原捕获 ELISA 试验检测及 Western blot分析。结果显示该重组弱毒株能在1~10代 BHK21细胞中稳定传代,扩增出约 3 000 bp片段,并经测序确证为基因 P12A3C;抗原捕获 ELISA 试验检测均为阳性;Western blot 分析表明转移载体 pUC119TKEGFPP7.5P12A3C 在感染的 GTPV AV41 BHK21 细胞中表达的蛋白可被 O型 FMDV 高免血清特异性识别,并具有反应原性。这些结果表明获得了表达 O 型口蹄疫病毒 P12A3C 基因的重组山羊痘弱毒株。

Abstract: The constructed gene EGFPP7.5P12A3C of FMDV O/China99 strain was linked to the linear vector by blunt end ligation using KpnⅠ enzyme site, and the recombinant vector pUC119TKEGFPP7.5P12A3C was obtained. Homologous recombination between recombinant vector with deleted gene TK and capripoxvirus attenuated strain took place in the cell BHK21. The recombinant attenuated strain was screened by choosing EGFP as marker gene. And the recombinant virus was identified by PCR, antigen capture ELISA and Western blot analysis. The recombinant attenuated strain can passage steady in the first to the tenth generation of BHK21 cell, a fragment of about 3 000 bp was amplified,and the gene was confirmed as P12A3C by sequencing. The results of antigen capture ELISA were all positive. Western blot analysis showed that the corresponding protein was expressed in transfer vector pUC119TK EGFPP7.5P12A3C infected GTPV AV41 BHK21 cells and it could be recognized by serotype O of FMDV serum. The result demonstrates that the recombinant attenuated CPV containing P12A3C gene of FMDV serotype O were obtained.