猪肠道病毒甲型3C基因的克隆和原核蛋白的表达

Cloning and prokaryotic expression of porcine enteriovirus A 3C gene

猪肠道病毒甲型(PEV A)是一种无囊膜、球形的单股正链RNA病毒,属于小RNA病毒科,能引起猪脑脊髓灰质炎、肠道、呼吸道、生殖器官疾病等多系统综合征。本试验采用基于PAS(PCR-based accurate synthesis)的方法,设计全长拼接引物,在引物的两端各设计了保护性碱基合成基因PEV A 3C,连入表达载体pCzn1;获得的重组质粒pCzn1-PEV A 3C转入Arctic Express表达菌株。诱导表达目的蛋白PEV A 3C,通过Ni柱亲和纯化获得目的蛋白。结果表明,成功构建了原核表达菌株,其所表达的融合蛋白分子质量约为21.7 ku,为下一步PEV A特异性ELISA检测方法的建立奠定了基础。

Porcine enteriovirus A（PEV A）is a capsule-free,single-stranded positive strand RNA virus,belonging to the small RNA virus family,and can cause porcine polio,intestinal,respiratory,genital diseases and other multiple system syndromes.In this study,the full-length splicing primers were designed based on PAS（PCR-based accurate synthesis）.The protective bases were designed at both ends of the primers.PEV A was synthesised and inserted into the expression vector pCzn1.The recombinant plasmid pCzn1-PEV A 3 C was transferred into Arctic Express expression strain.The target protein PEV A-3 C was induced and obtained by Ni affinity chromatography.The results showed that the recombinant protein was successfully expressed and the molecular weight with the fusion protein was about 21.7 ku,which laid the foundation for the establishment of PEV A specific ELISA method.