猪圆环病毒2型Cap蛋白的原核表达及其应用性研究

Prokaryotic Expression and Application of Cap Protein of Porcine Circovirus Type 2

本研究利用PCR扩增PCV2 ORF2基因,构建重组原核表达载体pET30a-ORF2,并通过酶切和测序鉴定,将测序正确的阳性重组菌接种至含卡那霉素的LB培养基中,经IPTG诱导表达,分别对表达产物进行SDS-PAGE分析及Western blot鉴定,并进行动物免疫以及攻毒试验评价其免疫原性及应用性。结果显示,蛋白分子量大小为37 kDa的PCV2 Cap基因在大肠杆菌中获得表达;免疫猪PCV2 Cap血清抗体ELISA滴度不低于1∶1600,免疫攻毒评价保护率为100%。猪圆环病毒2型Cap蛋白的成功表达,并具有良好的免疫原性,为研发更稳定、安全、高效的亚单位疫苗奠定了基础。

In this study,the complete PCV2 ORF2 gene was amplified by PCR and used to construct a prokaryotic expression vector pET30a-ORF2,which was confirmed by restriction enzyme digestion and sequencing.The recombinant bacteria with the vector pET30a-ORF2 were inoculated into LB medium containing kanamycin for expression of the Cap protein with induction by IPTG.The resulting product was examined in SDS-PAGE and visualized as molecular weight of 37 kDa in Western blot.Then,the immunogenicity and application were evaluated by animal immunization and challenge test.As a result,ELISA titer of serum samples from the immunized pigs was no less than 1:1600 and the protective rate was 100% against virulent challenge.The successful expression of PCV2 Cap protein and its good immunogenicity laid the solid foundation for further development of a safe,effective and inexpensive subunit vaccine and detection kit.