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  

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

作　　者：王建科[1] 张云德[1] 张强[1] 吴国华[1] 颜新敏[1] 朱海霞[1] 李健[1] 邵长春[1] 朱彩珠[1] 吴磊[1] 

机构地区：[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

出　　处：《畜牧兽医学报》2010年第4期434-440,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技基础条件平台项目(2005DKA21205-3)

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/Chi-na99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3000bp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 AV41BHK-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。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.

关 键 词：口蹄疫病毒 启动子P7.5 P1-2A-3C基因 羊痘病毒 

分 类 号：S852.659.6[农业科学—基础兽医学]