CAJ | 학술논문

[目的]为C型FMDV型特异性多克隆抗体、单克隆抗体的制备和FMDV定型提供理论依据.[方法]以含有C型口蹄疫病毒(FMDV)结构蛋白基因VP1的重组质粒pGEM-CP1为模板,设计特异性表达引物,扩增VP1及其C端编码区.对C型口蹄疫病毒VP1及其C端进行原核表达,并测定反应原性.利用纯化的C型VP1及其C端融合蛋白建立间接ELISA,分别对O、A、C、Asia 1四型豚鼠阳性血清进行检测,确定C型VP1及其C端与其他3型FMDV抗体的型间交叉反应性.[结果]构建了pPRO-CVP1、pPRO-CVP1c重组原核表达质粒,实现了C型口蹄疫病毒VP1及其C端的高效表达,目的蛋白的分子量大小分别为33 kD和20 kD.Western blot显示,VP1及其C端融合蛋白均可与对应血清型的豚鼠阳性血清反应.C型VP1及其C端与其他血清型的FMDV阳性血清均未发生交叉反应,且以VP1 C端的型特异性最好.[结论]获得了C型FMDV特异性抗原.
[목적]위C형FMDV형특이성다극륭항체、단극륭항체적제비화FMDV정형제공이론의거.[방법]이함유C형구제역병독(FMDV)결구단백기인VP1적중조질립pGEM-CP1위모판,설계특이성표체인물,확증VP1급기C단편마구.대C형구제역병독VP1급기C단진행원핵표체,병측정반응원성.이용순화적C형VP1급기C단융합단백건립간접ELISA,분별대O、A、C、Asia 1사형돈서양성혈청진행검측,학정C형VP1급기C단여기타3형FMDV항체적형간교차반응성.[결과]구건료pPRO-CVP1、pPRO-CVP1c중조원핵표체질립,실현료C형구제역병독VP1급기C단적고효표체,목적단백적분자량대소분별위33 kD화20 kD.Western blot현시,VP1급기C단융합단백균가여대응혈청형적돈서양성혈청반응.C형VP1급기C단여기타혈청형적FMDV양성혈청균미발생교차반응,차이VP1 C단적형특이성최호.[결론]획득료C형FMDV특이성항원.
[Objective]The aim was to provide a theoretical basis for preparation of polyclonal antibodies and monoclonal antibody that of type specificity, as well as Foot-and-mouth Disease Virus (FMDV) typing. [Method] The recombinant plasmid pGEM-CP1 that contained VP1 gene of FMDV of serotype C was used as template for the VP1 and its C terminus coding fragments of FMDV of serotypes C amplification. The coding fragments of VP1 and its C terminus were respectively cloned into prokaryotic expression vector for prokaryotic expression and the reactionogenicity was detected. The purified fusion protein of FMDV VP1 and its C terminus of serotype C were used to construct the indirect ELISA method to detect positive sera of four serotypes A, O, C and Asia 1 of guinea pig and determine the cross reactivity of FMDV antibody of VP1 and its C terminus of serotype C with other three serotypes. [Result]The recombinant prokaryotic expression plasmids of pPRO-CVP1 and pPRO-CVP1c were constructed, FMDV VP1 and its C terminus of serotype C were expressed in high level, and the molecular weight of target proteins was 33 kD and 20 kD respectively. Western blot result showed that the fusion protein of VP1 and its C terminus could react with the positive sera of guinea pig of the same serotype. ELISA results revealed that VP1 and its C terminus of serotype C are type-specific and no cross-reactivities were shown between guinea pig positive sera of FMDV of serotype C with the other three serotypes, and the C terminus showed better type-specificity. [Conclusion] FMDV specific antigen of serotype C was obtained.