马动脉炎病毒GL蛋白主要抗原域的表达及间接ELISA的初步建立  

Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay

作　　者：梁成珠[1] 曹瑞兵[1] 魏建超[1] 朱来华[1] 陈溥言[1] 高宏伟 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,南京210095 [2]青岛出入境检验检疫局,青岛266002

出　　处：《微生物学报》2006年第3期436-440,共5页Acta Microbiologica Sinica

基　　金：国家质量监督检验检疫总局科研资助项目(2003-IK-005)~~

摘　　要：在分析马动脉炎病毒GL蛋白抗原性的基础上,设计一对引物克隆GL蛋白一段抗原性较好的抗原域编码基因。将克隆的基因插入pET-32a的BamHⅠ和XhoⅠ之间构建了GL蛋白主要抗原域原核表达载体pET-GL1。将pET-GL1质粒转化BL(21)宿主菌后,对培养和表达条件进行了优化,实现了EAV GL蛋白主要抗原域的高效表达。免疫印迹试验表明获得的表达产物具有良好的反应原性。应用His.Bind亲和层析柱纯化重组EAV-GL1蛋白,以纯化的重组GL1蛋白作为检测抗原,初步建立了检测马动脉炎病毒抗体的iGL1-ELISA。结果表明,抗原的最佳包被浓度为9.65μg/mL,血清的最佳稀释度为1∶80,阳性标准初步定为:待检血清OD490>0.4,且待检血清OD490/阴性血清OD490>2。应用iGL1-ELISA对马血清样品进行检测,结果表明iGL1-ELISA与病毒中和试验的符合率达到94.1%,与国外同类试剂盒的符合率达到95.6%。According to the antigenic analysis of equine arteritis virus （EAV） GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH Ⅰ and Xho Ⅰ and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21（DE3） and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was confirmed by Western blot. The recombinant GL1 protein was purified by the means of His. Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against EAV with the purified GL1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 9.65μg/mL and the optimal dilution of serum was 1： 80. The positive criterion of this ELISA assay is ODthetesmdserurn 〉 0.4 and ODthetested serum/ODthe negative serum 〉 2.0, The iGL-ELISA was evaluated versus micro-virus neutralization test. The ELISA was performed on 900 sera from which were preserved by this lab during horse entry/exit inspection, the agreement （94.1% ） of these test were considered suitable for individual serological detection. In another test which 180 sera samples were detected by iGL-ELISA and INGEZIM ELISA kit respectively. The agreement ratio between the two methods is 95.6%.

关 键 词：马动脉炎病毒 GL蛋白 主要抗原域 原核表达 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]