狂犬病病毒糖蛋白胞外区基因的原核表达、纯化及鉴定

Prokaryotic Expression,Purification and Identification of Rabies Virus Glycoprotein Extracellular Domain Gene

利用RT-PCR技术,将狂犬病病毒株ERA株糖蛋白胞外区基因分两段进行扩增,经克隆与序列分析,两片段大小分别为523,381 bp,各编码174,127个氨基酸,分子量分别为19.7,14.5 kDa。在原核表达载体pET-43a中分别克隆了这两段糖蛋白基因,将重组质粒转化到表达菌BL21(DE3)中,经SDS-PAGE电泳分析,在分子量约为94,89kDa处分别出现新蛋白带,和预期的目的蛋白的分子量相符。两重组蛋白主要以可溶形式表达,利用Ni2+亲和层析柱纯化了蛋白,纯度大于95%。Western-Blot分析结果显示,两种重组融合蛋白均可与兔抗RBV多抗血清反应,具有良好的反应原性。

The glycoprotein genes of extracellular domain were amplified from rabies virus ERA strain by RTPCR,The products were respectively cloned and sequenced.The result showed that the two fragments length were composed of 523,381 bp,which encoded 174,127 amino acids respectively.It＇s molecular weight were 19.7,14.5 kDa.The two products of glycoprotein gene were sub-cloned into the prokaryotic expression vector pET-43a.The positive recombinants were transformed into E.coli BL21（DE3）,SDS-PAGE showed that the proteins were highly expressed in E.coli and the molecular weight were 94,89 kDa.The results are consistent with the expected molecular weight of interest proteins.The recombinant proteins were mainly expressed in soluble form and purified by Nichela-ting chromatography.The results of Western-Blot analysis revealed that the two expressed proteins were recognized specifically by rabbit antiRBV polyclonal antibody,The stable expression of the proteins and the analysis of its anti-genic specificity provide the foundation to develop the ELISA diagnostic kit for rabies.