鸭瘟病毒gD基因胞外区的原核表达及ELISA检测方法的建立与应用

Prokaryotic expression of extracellular region of gD gene of duck plague virus and establishment,application of the recombinant gD-based enzyme-linked immunosorbent assay

为研究分离鉴定的鸭瘟病毒gD基因(GenBank登录号:EU195085)及其编码蛋白的应用,采用PCR方法从鸭瘟病毒CHv毒株DNA中获取编码gD胞外区基因729bp片段,构建重组表达载体pET32a-gD,转化受体菌E.coli BL21(DE3),IPTG诱导表达重组gD蛋白。结果显示,该重组蛋白分子质量约为31ku,为gD胞外区与载体6个组氨酸的融合表达产物,与gD胞外区预期大小一致(27.5ku);经离心收集菌体,超声裂解后,SDS-PAGE分析结果显示,gD蛋白主要以包涵体形式表达;将包涵体溶解后进行Ni-NTA亲和层析可较好地纯化重组蛋白。Western-blot检测显示,gD蛋白能与兔抗鸭瘟病毒血清特异结合,具有较好的免疫反应性。利用该蛋白建立了ELISA检测方法,优化了该方法的各个反应条件,并进行了初步应用,为该方法的临床应用奠定了基础。

In order to get the gD gene（GenBank：EU195085）of duck plague virus（DPV）CHv strain, according to the bioinformatics analysis information,extracellular region of gD gene fragment about 729 bp was amplified by PCR method and cloned into an expression vector pET-32a to construct recombinant expression vectors pET32a-gD. The recombinant expression vectors were then transformed into the compe tent host Escherichia coli BL21（DE3） with the expression of recombinant extracellular region of gD induced by IPTG. The molecular weight of fusion protein was corresponding to the estimated molecular weight of extracellular region of gD protein（27.5 ku）, and the extracellular region of gD was mainly expressed as inclusion body. Western-blot analysis demonstrated intense gD immunoreactivity with rabbit anti-DPV serum. And a gD-based enzyme-linked immunosorbent assay（ELISA） was established,and testedwith 90 field samples. The research lays foundation to apply the ELISA in field.