小反刍兽疫病毒核衣壳蛋白抗原性与特异性的比较

为筛选建立小反刍兽疫病毒N蛋白双抗原夹心ELISA抗体检测方法的抗原原料,本研究通过优化大肠杆菌密码子、优化蛋白表达与纯化条件等方法,分别在pET-30a与pET-32a两个不同原核表达载体中成功获得了可溶性的小反刍兽疫核衣壳蛋白(N)。分别对重组大肠埃希氏菌pET-30a-(N)-BL21(DE3)株与pET-32a-(N)-BL21(DE3)株种子批的P7代与P20代进行蛋白诱导表达与抗原提取纯化,共获得6批小反刍兽疫病毒pET-30a-(N)蛋白与pET-32a-(N)蛋白,并对蛋白的浓度、纯度、抗原性和特异性进行检验。结果表明两个纯化后的重组抗原与羊小反刍兽疫病毒阳性血清有明显反应,具有较好的反应性,而与羊痘病毒阳性血清、羊口蹄疫病毒阳性血清、山羊关节炎/脑炎病毒阳性血清等特异性血清均无交叉反应,具有较好的特异性。本研究结果为今后以N蛋白为基础建立相应的抗原捕获ELISA方法,竞争ELISA检测方法、间接ELISA检测方法以及双抗原夹心ELISA抗体检测方法打下了坚实的基础。

Comparative study on the antigenicity and specificity of nucleocapsid protein of Peste des Petits Ruminants virus

To screen and establish the antigenic material for the detection of the N protein double antigen sandwich ELISA antibody of the Peste des Petits Ruminants virus,the E.coli codons,protein expression and purification conditionswere optimized,and the soluble Peste des Petits Ruminants nucleocapsid protein(N)was successfully obtained in two different prokaryotic expression vectors of pET-30 a and pET-32 a.After protein-induced expression and purification of the seed batches of the recombinant pET-30 a-(N)-BL21(DE3)strain and the pET-32 a-(N)-BL21(DE3)strain of the P7 and P20 generations,a total of 6 batches of the pET-30 a-(N)and pET-32 a-(N)proteins of the Peste des Petits Ruminants virus were obtained,and the concentration,purity,antigenicity and specificity of the proteins were tested.The results showed that the two purified recombinant antigens had obvious reaction with the positive serum of the Peste des Petits Ruminants virus,but no cross reaction with the positive sera of the sheep pox virus,sheep foot-and-mouth disease virus and goat arthritis/encephalitis virus.These two antigens had significant sensitivity and specificity.The results of this study laid a foundation for the establishment of competitive ELISA assay,indirect ELISA assay and double antigen sandwich ELISA antibody assay,based on the N protein.