【摘要】 为了研究鸭甲肝病毒3型(DHAV-3)VP1蛋白的优势抗原区,试验根据DHAV-3 VP1基因序列设计4对特异性表达引物,用PCR获得VP1-a~d基因片段,并在p ET-30a(+)/Rosetta(DE3)p LysS系统中进行原核表达;经IPTG诱导重组蛋白获得表达,用Ni-NTA柱亲和层析获得纯化的重组蛋白;同时以DHAV-3病毒为免疫原制备鼠抗DHAV-3多克隆抗体,通过I-ELISA和Westernblot初步鉴定。结果表明:p ET-30a-VP1-a、p ET-30a-VP1-b、p ET-30a-VP1-c和p ET-30a-VP1-d与鼠抗DHAV-3多克隆抗体都发生反应,抗原量相同的情况下VP1-c和VP1-d段显色较VP1-a和VP1-b深,说明这两段反应性相对较强;4种截短蛋白与鸭抗DHAV-3阳性血清进行反应,只有p ET-30a-VP1-c显色。说明VP1-c免疫原性较强,DHAV-3 VP1蛋白的90~175 aa为其优势抗原区。更多还原

【Abstract】 In order to research the dominant epitope region of DHAV-3 VP1,according to the sequence of DHAV-3 VP1 gene,four pairs of specific primers were designed and the VP1-a ~ d gene fragments were cloned by PCR. The recombinant VP1-a ~ d proteins were produced in prokaryotic expression system( p ET-30a( +)/Rosetta( DE3) p Lys S. The recombinant proteins were induced by IPTG and purified by Ni-NTA column affinity chromatography. Meanwhile,DHAV-3 was used as immunogen to prepare mouse anti-DHAV-3 serum. The results of I-ELISA and Western-blot showed that p ET-30a-VP1-a ~ d reacted with mouse anti-DHAV-3 serum. At the same dose of antigen,the color of the band of VP1-c and VP1-d were deeper than VP1-a and VP1-b,which indicated that the former epitope region manifested the better immunogenicity. Four recombinant proteins were interacted with duck anti-DHAV-3 positive serum,only p ET-30a-VP1-c showed the color of band.It was concluded that the VP1-c manifested better immunogenicity than others,and 90 ~ 175 aa was the dominant epitope region of DHAV-3VP1.更多还原