传染性法氏囊病病毒超强毒株衣壳蛋白的可溶性表达、纯化与活性分析

Soluble Expression,Purification and Activity Analysis of Capsid Protein of Very Virulent Infectious Bursal Disease Virus

为获得高质量的病毒衣壳蛋白VP2,本研究克隆了传染性法氏囊病病毒(infectious bursal disease virus,IBDV)超强毒株Gx的VP2基因,并亚克隆至载体pCold-Ⅰ,进而获得重组原核表达质粒pCold-Ⅰ-GxVP2,在Transetta(DE3)工程菌中优化条件进行IBDV衣壳蛋白VP2的可溶性表达,运用Ni-NTA亲和层析和凝胶过滤两种技术串联的方法进行蛋白纯化,运用单克隆抗体介导的Western blotting技术鉴定纯化蛋白的特异性,免疫SPF鸡鉴定纯化蛋白的免疫活性。结果显示,在冷休克条件下,IBDV衣壳蛋白VP2在Transetta(DE3)工程菌中实现了可溶性表达;通过纯化获得了高纯度的VP2,浓度为542μg/mL;该蛋白不仅能与VP2单克隆抗体特异性反应,还能刺激鸡产生特异性免疫应答,具有良好的免疫活性。本研究在IPTG为1mmol/L,15℃、120r/min,诱导时间为24h的条件下,实现了有免疫活性的IBDV衣壳蛋白VP2的可溶性表达和纯化。高纯度、可溶、具有功能活性的衣壳蛋白的制备,为深入开展IBDV致病机制研究奠定了基础。

To obtain the capsid VP2 with high quality,VP2 gene of very virulent infectious bursal disease virus（vvIBDV）Gx was cloned and inserted into pCold-Ⅰ and the prokaryotic expression plasmid pCold-Ⅰ-GxVP2 was constructed.In engineering bacteria Transetta（DE3）,the induction conditions of protein VP2 expression were optimized.With affinity chromatography and gel filtration,protein VP2 was purified.With the monoclonal antibody directed Western blotting,protein VP2 was identified.Using SPF chicken,immunocompetence of VP2 was evaluated.The results showed that the dissoluble protein VP2 was expressed successfully in Transetta（DE3）incold-shock conditions;Protein VP2 was purified and the concentration was 542μg/mL;The purified protein VP2 not only reacted with the monoclonal antibody against protein VP2,but also induced specific immune response in immunized chickens.In general,with 15 ℃ of cold-shock condition,120r/min of shaking culture,1mmol/L of IPTG,inducting for 24 h,soluble capsid VP2 of IBDV with immunocompetence was successfully expressed and purified.The preparation of highly purified,soluble capsid protein with functional activity laid the foundation for further researches on the pathogenic mechanism.