猪瘟病毒复合多表位抗原基因的克隆表达及其免疫学特性

Cloning,expression and immunological characteristics of compound multi-epitope gene of classical swine fever virus

为研究猪瘟病毒多表位疫苗,参照GenBank及文献中已发表的猪瘟病毒抗原表位,结合计算机分析软件进行优化和组合。将重组的多表位基因BT23人工合成克隆至pMD18-T质粒中,利用BamHⅠ和EcoRⅠ双酶切并回收BT23基因,将BT23基因片段亚克隆插入pGEX-6P-1表达载体中,构建了猪瘟病毒复合多表位抗原基因的原核表达质粒pGEX-BT23,经酶切和测序鉴定正确后转化感受态细胞BL21(DE3),并进行了IPTG诱导表达。经SDS-PAGE分析,以终浓度为0.9mmol/L的IPTG进行诱导,8h后表达量最高,表达产物为融合蛋白,并且以包涵体形式存在,分子质量约36ku,表达产量约占菌体总蛋白的30%。Western-blot检测结果表明,表达的融合蛋白能与猪瘟病毒阳性血清发生特异性反应,说明表达产物具有良好的反应原性。免疫攻毒试验结果表明,复合多表位融合蛋白具有免疫保护作用,这为应用该融合蛋白制备猪瘟病毒免疫血清学诊断试剂和多表位疫苗的研究奠定了基础。

To develop multi-epitope vaccine against classical swine fever, a multi-epitope gene BT23 was optimized and artificially synthesized according to the epitope to sequence of classical swine fever virus（CSFV） in GenBank and literatures,and was then cloned into the pMD18-T vector. After BamHⅠ and EcoRⅠ double digestion,BT23 gene was recovered and subcloned into the pGEX-6P-1 vector to form a recombinant plasmid pGEX-BT23. And then the BT23 gene in pGEX-BT23 was expressed in Escherichia coli BL21（DE3） competent cells. The highest expressed fusion protein was 30% of total bacterial protein after being induced with IPTG at 0.9mmol/L for 8h in a inclusion body manner with molecular weight of 36ku as certified by SDS-PAGE. Western-blot analysis showed that the expressed product had good reactinogenicity,which specially reacted with CSFV positive sera. Moreover,the fusion protein could induce an efficient immune protection against HCLV infection,which provide foundations for the preparation of the CSFV serological diagnostic reagents and for the development of multi-epitope vaccine.