A型口蹄疫病毒抗原表位与牛IgG重链恒定区的融合表达及活性鉴定

Fusion Expression and Activity Identification of Type A FMDV Antigenic Epitope with Heavy Chain Constant Region of Bovine IgG

为了研制广谱高效的A型口蹄疫新型多表位疫苗,根据GenBank数据库的A型代表毒株VP1序列设计并合成了VP1结构蛋白上的主要抗原表位DNA段,即135aa～160aa和200aa～213aa,与牛IgG重链恒定区编码基因连接,将合成的2个表位基因经XhoⅠ、EcoRⅠ和BamHⅠ酶切后依次克隆到pET-30a(+)载体上,构建重组质粒pRE2IgG,将其转化到大肠埃希菌BL21(DE3)感受态细胞,以IPTG诱导表达融合蛋白pRE2IgG,对表达产物进行SDS-PAGE电泳分析,Western blot检测。结果显示,重组蛋白获得高效表达,并以包涵体形式存在,其分子质量约为60ku,且能与抗A型FMDV阳性血清发生特异性反应,具有良好的反应活性。

In order to develop a new broad-spectrum multi-epitope vaccine of type A foot-and-mouth disease virus（FMDV）,one DNA fragment encoding VP1main epitope 135aa-160aa and 200aa-213aa was designed and synthesized according with the VP1gene sequences of six representative FMDV type A strains from GenBank database,and then attached to the gene of heavy chain constant region of bovine IgG.Via using the XhoⅠ,EcoRⅠand BamHⅠsites,both were cloned into the pET-30a（＋）vector to form the recombinant plasmid pRE2IgG,with the gene of heavy chain constant region of bovine IgG.By using the BamHⅠ,EcoRⅠand XhoⅠsites,both of the two genes were cloned into the pET-30a（＋）vector to form the recombinant plasmid pRE2IgG,and then transformed the recombinant plasmid into E.coli BL21（DE3）competent cells.SDS-PAGE and Western Blot results indicated that the fusion protein pET30a105106 IgG was highly expressed in the form of inclusion bodies in E.coli induced by IPTG,and its molecular weight is about 60ku,and can react with FMDV type A positive serum.