鸭肠炎病毒NP蛋白的原核表达及间接ELISA方法的建立

Prokaryotic expression of nucleocapsid protein of duck enteritis virus and development of indirect ELISA

设计一对特异性引物扩增出鸭肠炎病毒(DEV)核衣壳蛋白(NP)基因,并将其定向插入到原核表达载体pET32a上,构建了NP基因的原核表达载体pET-NP;将重组载体pET-NP转化表达宿主菌BL21后,经SDS-PAGE分离后行Western blot显示,获得的表达产物具有良好的免疫原性;应用His.Bind亲和层析柱纯化重组NP蛋白,并以此作为包被抗原,初步建立了检测鸭肠炎病毒抗体的iNP-ELISA;经方阵滴定确定,重组蛋白抗原的最佳包被浓度为5.0μg/L,血清最佳稀释度为1∶80,阳性判定标准为:待检血清OD405值≥1.2,且待检血清OD405和阴性血清OD405的比值≥2.0;应用iNP-ELISA对450份鸭血清样本进行检测,结果iNP-ELISA与全病毒包被的iDEV-ELISA符合率达90.9%。

A pair of primers was designed to amplify the nuleocapsid protein （NP） gene from duck enteritis virus （DEV） genome. The cloned fragment was digested with BamH I and Hind Ⅲ, and then inserted into pET32a vector to obtain the recombinant pET-NP plasmid. The recombinant plasmid was transformed into E. coli BL21, and expressed in a high level after induced with IPTG. The SDS-PAGE and Western blotting analysis of expressed product indicated that the fusion protein was about 48 ku in molecular weight and showed specific im- munoreactivity with anti-DEV sera. The recombinant protein was purified with His. Bind resin protein purification procedure, and then iNP- ELISA was established using the purified protein as antigen. The optimal concentration of the coated antigen was 5.0 μg/mL and the optimal dilution of serum was 1 ： 80. The positive criterion of this ELISA assay was OD405 ≥ 1.2 in the tested serum and OD405 （ tested serum） / OD403 （negative serum） I〉2. O. Four hundreds and fifty serum samples from Guizhou province were detected by iNP-ELISA and iDEV- ELISA with DEV as the coating antigen, respectively, and the agreement ratio between the two methods was up to 90. 9%.