检测猪繁殖与呼吸综合征抗体的重组N蛋白-ELISA的建立

Establishivient of Recombinant N Protein Based ELISA for Detection of Antibody Against Porcine Reproductive and Respiratory Syndrome Virus

将已构建成功的重组质粒pETN转化入E.coliBL21(DE3)中,在最佳诱导条件下获得重组N蛋白。随后用His-Bind对表达产物进行纯化,对纯化效果及纯化产物的特异性分别用SDS-PAGE电泳及Western-blot试验检测。在此基础上,以纯化的重组N蛋白作为包被抗原,对各种条件进行优化(如抗原的包被,作用时间及底物的选择),确定了判定标准,建立了检测PRRS抗体的间接ELISA方法。用此方法检测了200份血清样品,并与IDEXX公司ELISA试剂盒检测结果相比较,符合率达91%。

The recombinant plasmid pETN was transformed into E.coli BL21 (DE3) host cell and the expression product-recombinant nucleocapsid protein of PRRSV was obtained under the optimized condition of host cell cultivation and IPTG induction. Consequently, the expression product was purified by means of his-binding resin protein purification procedure. SDS-PAGE and Western-blot were used to detect the purification effect and the specificity of purified recombinant nucleocapsid protein of PRSSV. The purified N protein was used to coat 96-well plate, each step was optimized, such as coating concentration of recombinant nucleocapsid protein, scample dilutim, chromogen (TMB) and stop solution as well as concentration. As a result, an indirect ELISA was established to detect antibody against PRRSV. About 200 serum samples were detected by the method and IDEXX ELISA kit, respectively. The agreement ratio between the two method reached at 91%.