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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 Enterovirus (PEV A) is a non-enveloped, spherical, single-stranded positive-sense RNA virus belonging to the family of picornaviruses that causes polio, porcine, respiratory, and reproductive disorders Systemic syndrome. In this study, PAS (PCR-based accurate synthesis) -based method was used to design a full-length splicing primer. PEV A 3C was designed at both ends of the primer and inserted into the expression vector pCzn1. The obtained recombinant plasmid pCzn1 -PEV A 3C into Arctic Express expression strain. The target protein PEV A 3C was induced and expressed by Ni column affinity purification. The results showed that the prokaryotic expression strain was successfully constructed and the molecular weight of the expressed fusion protein was about 21.7 ku, which lays the foundation for the establishment of the PEV A-specific ELISA assay.