猪圆环4型病毒Cap蛋白的原核表达

Prokaryotic expression of porcine circovirus type 4 cap protein

以本实验室扩增的内蒙猪圆环4型病毒(PCV4)毒株的ORF2基因序列为参考,对其密码子进行优化并送往公司进行质粒合成,通过设计特异性引物,扩增出Cap蛋白基因。将目的基因克隆至pET-28a载体,构建出pET28a-PCV4-Cap重组表达质粒。将重组质粒转化至表达宿主菌BL21(DE3)pLysS中进行诱导表达,制备PCV4 Cap重组蛋白。优化表达条件和SDS-PAGE分析结果显示,Cap重组蛋白能够在沉淀中大量表达且在IPTG终浓度为0.2 mmol/L、37℃诱导6 h条件下表达量最高,其约为27 kDa。经Western blot验证,条带单一,与预期结果相同。本试验结果为了解PCV4 Cap蛋白及建立PCV4血清流行病学快速检测方法奠定了基础。

The ORF2 gene sequence of Inner Mongolian PCV4 strain was used as a reference.By designing specific primers,the gene was amplified and cloned into pET28 a vector and the recombinant expression plasmid pET28 a-PCV4-Cap was constructed.The pET28 a-PCV4-Cap was transformed into the expressing host bacterium BL21(DE3)pLysS to express the recombinant Cap protein.By optimizing the expression conditions,SDS-PAGE analysis showed that recombinant Cap protein could be expressed in large quantities in the precipitation and the highest expression level was achieved when the final concentration of IPTG was 0.2 mmol/L and the expression was induced for 6 h at 37℃,its size was about 27 kDa.Western blot result showed that the band was specific,it was consistent with the expected results.PCV4 unique structural protein Cap was expressed,laying a foundation for understanding PCV4 Cap protein and establishing a rapid detection method for PCV4.