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
표체O형구제역병독P1-2A-3C기인적산양두병독약독주적구건급사선
Construction and Screening of the Recombinant Attenuated CPV Expressing P1-2A-3C Gene of FMDV Serotype O
  • 万方数据
    畜牧兽医学报 축목수의학보

    2010年 4期 434-440 ,共7页

    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

    왕건과%장운덕%장강%오국화%안신민%주해하%리건%소장춘%주채주%오뢰

    口蹄疫病毒%启动子P7.5%P1-2A-3C基因%羊痘病毒 口蹄疫病毒%啟動子P7.5%P1-2A-3C基因%羊痘病毒
    구제역병독%계동자P7.5%P1-2A-3C기인%양두병독
    FMDV%promotor P7.5%P1-2A- 3C gene%CPV
    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K pn I酶切后的线性载体pUC119-TK中,得到重组载体pUC119-TK-EGFPLP7.5-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.
    위구건화사선표체O형구제역병독P1-2A-3C기인적산양두병독약독주,용이구건적구제역병독O/Chi-na99독주적EGFP-P7.5-P1-2A-3C기인정체통과평말단련접도K pn I매절후적선성재체pUC119-TK중,득도중조재체pUC119-TK-EGFPLP7.5-P1-2A-3C.중조재체통과결실적TK기인여양두병독약독주재BHK-21세포중동원중조,용EGFP작위표기사선출중조독주,병진행PCR감정、항원포획ELISA시험검측급Western blot분석.결과현시해중조약독주능재1～10대BHK-21세포중은정전대,확증출약3 000 bp편단,병경측서학증위기인P1-2A-3C;항원포획ELISA시험검측균위양성;Western blot분석표명전이재체pUC119-TK-EGFP-P7.5-P1-2A-3C재감염적GTPV AV41 BHK-21세포중표체적단백가피O형FMDV고면혈청특이성식별,병구유반응원성.저사결과표명획득료표체O형구제역병독P1-2A-3C기인적중조산양두약독주.
    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 Kpn I 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.
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