猪瘟病毒多表位基因的表达及其免疫原性分析

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

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0.9mmol.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43ku,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。

Genetical engineer T, B cell epitopes of Classical swine fever virus （CSFV） were devel- oped by employing molecular biological tools, and their immunogenicity has been primarily evalu- ated by immunizing experimental animals. A DNA fragment which encoding a tandem repeat pro- tein of T, B cell epitopes of CSFV were designed and chemically synthesized, and it was cloned into pGEX-6P-1 vector in turn to {orm a recombinant plasmid pGEX-BT500. A chimeric protein was obtained after transforming the pGEX-BTS00 into Escherichia coZi 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 recom- binant 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 re- combinant protein is a potential multiepitope peptide vaccine candidate to prevent CSFV infection.