Asia1型口蹄疫病毒多表位基因的原核表达及ELISA检测方法的建立  

Prokaryotic expression of multi-epitope gene of foot-and-mouth disease virus serotype Asia 1 and establishment of an ELISA for detection of the virus

作　　者：鲁会军[1] 金宁一[1] 郑敏[1] 韩松[1] 霍晓伟[1,2] 田明尧[1] 李霄[1] 金扩世[1] 

机构地区：[1]中国人民解放军军事医学科学院军事兽医研究所,吉林长春130062 [2]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医科学》2009年第1期45-49,共5页Chinese Veterinary Science

基　　金：国家“十一五”科技支撑计划项目(2007BAD55B05)

摘　　要：根据国内流行的Asia 1型口蹄疫病毒的基因组特性,合成了Asia 1型口蹄疫病毒2个流行毒株VP1基因的5个抗原表位,并将其克隆到pMD18-T载体上,再按设计的酶切位点连接,构建出Asia 1型口蹄疫病毒VP1双拷贝基因片段(VP1 Asia)。然后,将VP1 Asia基因连接到原核表达载体pET-28a(+)上,构建了重组表达质粒pET-28a-VP1 Asia,接着将其转入BL21菌中进行原核表达。以纯化的表达蛋白作为抗原检测了牛Asia 1型口蹄疫病毒阳性血清。结果显示,VP1 Asia基因在大肠杆菌中获得了高效表达,以纯化的VP1Asia蛋白作为抗原建立了检测Asia 1型口蹄疫病毒抗体的ELISA方法。以建立的ELISA方法和标准Asia 1型口蹄疫液相阻断ELISA试剂盒对30份临床样品进行了检测,两种检测方法的阳性率分别为90.0%(27/30)和93.3%(28/30),符合率为89.7%(26/29)。本研究为组装Asia 1型口蹄疫病毒抗体ELISA诊断试剂盒奠定了基础。Five epitopes of VP1 of two strains of foot-and-mouth disease virus（FMDV） serotype Asia 1 were synthesized,and cloned into the pMD18-T vector. According to the designed restriction enzyme sites, the double copy fragment was constructed,and then designated VPlAsia. The VPlAsia gene was cloned in to the prokaryotic expression vector pET-28a （q＋）, and the recombinant plasmid pET-28a-VP1Asia was constructed. The recombinant plasmid was transformed into BL21 （DE3） competent ceils, and SDS-PAGE analysis showed that the VPlAsia gene was highly expressed. The recombinant proteins were purified by the washing of inclusion bodies and gel extraction. The VP1Asia protein was used as the diagnostic antigen to detect the bovine positive sera of FMDV serotype Asia 1. The VPlAsia protein and the FMDV serotype Asia 1 liquid-phase blocking ELISA Kit were used to detect 30 samples. The results showed that the positive ratio was 90.0 % and 9 3.3 %, respectively, and the coincidence was 8 9.7 %（2 6 / 2 9） between the two methods.

关 键 词：口蹄疫 亚洲1型 多表位基因 酶联免疫吸附试验 

分 类 号：S852.659.6[农业科学—基础兽医学] R446.61[农业科学—兽医学]