传染性法氏囊病毒VP2蛋白的原核表达及其免疫效果评价

Prokaryotic Expression of VP2 Protein of Infectious Bursal Disease Virus and Its Immunization Evaluation

为提高原核表达传染性法氏囊病毒(IBDV)VP2蛋白纯度,在大肠杆菌中表达VP2蛋白,并对其进行纯化方法优化及其免疫原性分析。利用琼脂扩散试验(AGP)测定VP2蛋白效价并进行SDS-PAGE和Western blot鉴定;采用饱和硫酸铵溶液和离子交换层析对目的蛋白进行纯化,并对纯化填料、洗脱液离子强度及缓冲液的pH值进行优化,以确定最佳纯化条件。将纯化的VP2蛋白免疫SPF鸡,定期采血用ELISA方法测定其免疫抗体并进行病理组织检测。SDS-PAGE及Western blot鉴定结果显示,VP2分子质量约为50 ku,且具有良好反应原性,VP2蛋白可溶性表达的AGP效价为1∶16。纯化结果显示,利用强阴离子层析优化盐离子浓度及pH值可获得纯度较高的VP2蛋白,其质量浓度为0.6 mg/mL。纯化的VP2蛋白加商业佐剂免疫鸡28 d后,鸡产生的特异性免疫抗体滴度最高,且攻毒后的保护率可达到80%,法氏囊无明显病理组织损伤。综上,利用大肠杆菌成功表达了VP2蛋白,纯化的VP2蛋白纯度较高且具有良好的免疫原性。

In order to improve the purity of infectious bursal disease virus(IBDV)VP2 protein,this study expressed VP2 protein in Escherichia coli,optimized purification methods and estimated its immunogenicity.VP2 protein was identified by SDS-PAGE and Western blot.The titer of VP2 protein was evaluated by the agar gel immunodiffusion test(AGP).To determine the optimal purification conditions,the target protein was purified by saturated ammonium sulfate solution and ion exchange chromatography,and various steps in proteins purification were optimized to determine the optimal purification conditions including the purified packing,eluent concentration and pH value of the buffer.Then,SPF chickens were immunized with the purified VP2 protein,and the immunogenicity of VP2 protein was determined with ELISA by detecting the periodically collected blood.The results showed that VP2 protein was 50 ku and had good reactogenicity by SDS-PAGE and Western blot,and the titer of VP2 protein determined by AGP was 1∶16.Using a strong anion column to optimize the salt ion concentration and pH value,a higher purity VP2 protein could be obtained with concentration of 0.6 mg/mL.Twenty-eight days after immunized with purified VP2 protein and commercial adjuvant,the titer of specific antibodies was the highest,and the protection rate could reach 80%.No obvious histopathological damage was found in the bursal.Above all,VP2 protein was successfully expressed in Escherichia coli,and the purification method was optimized.The purified VP2 protein had high purity and good immunogenicity.