猪瘟病毒多表位基因的表达及其免疫原性分析

体外表达猪瘟病毒(CFSV)T、B细胞表位,并对表达产物的免疫原性进行分析.人工合成CFSV的多个T、B细胞表位及表位之间的连接linker基因,并将该基因插入pGEX-6P-1表达载体,经酶切和测序鉴定获得重组阳性克隆,成功构建了CFSV复合多表位抗原基因的原核表达质粒pGEX-BT500,转化E.coli BL21,IPTG诱导表达,纯化表达产物并免疫兔.结果:通过IPTG诱导目的基因可高效表达,SDS-PAGE结果表明,以终浓度为0.9 mmol· L-1的IPTG进行诱导,7h后表达量最高,产物分泌表达,相对分子质量约43 ku,表达产量约占菌体总蛋白的30％.Western blotting检测表明,表达的融合蛋白能与猪瘟阳性血清发生特异性反应；兔攻毒试验表明所免疫表达产物可保护(根据发热反应评价)兔.结果表明表达获得的产物具有良好的反应原性,这为应用该融合蛋白制备CSFV免疫血清学诊断试剂和多表位疫苗研究奠定了基础.

Expression of Compound Multi-epitope Gene of Classical Swine Fever Virus in E.coli and the Assessment of Its Immunologicity

Genetical engineer T,B cell epitopes of Classical swine fever virus(CSFV) were developed by employing molecular biological tools,and their immunogenicity has been primarily evaluated by immunizing experimental animals.A DNA fragment which encoding a tandem repeat protein of T,B cell epitopes of CSFV were designed and chemically synthesized,and it was cloned into pGEX-6P-1 vector in turn to form a recombinant plasmid pGEX-BT500.A chimeric protein was obtained after transforming the pGEX-BT500 into Escherichia coli BL21(DE3) host cell and induced by IPTG.The western blotting analysis showed that the expressed products have a good reactogenicity,which can react specially with CSF positive sera.Inoculation with 100 μg recombinant protein induced strong neutralizing antibody response in rabbits.Our study indicated that the expressed T,B epitopes can act as the carrier protein for CSFV peptide epitopes,and this recombinant protein is a potential multiepitope peptide vaccine candidate to prevent CSFV infection.