蓝舌病1型病毒VP2蛋白的原核表达及免疫反应性分析

摘要

试验旨在研究蓝舌病1型病毒(bluetongue virus serotype 1, BTV-1)VP2蛋白体外表达产物免疫反应性.根据已发表的BTV-1 Y863毒株L2基因序列设计合成特异性BTV-1 PCR引物,通过RT-PCR方法扩增L2基因,将纯化的L2基因克隆至pEASY-Blunt E1表达载体,对重组质粒进行鉴定,将阳性重组质粒L2克隆至大肠杆菌BL21(DE3)感受态细胞进行表达,对获得的纯化的BTV-1 VP2重组蛋白进行Western blotting、ELISA、阻断ELISA分析.结果显示,BTV-1 VP2蛋白在pEASY-Blunt E1 载体上以包涵体形式表达,通过Ni-NTA亲和层析,160和200 mmol/L咪唑是洗脱BTV-1 VP2蛋白表达产物的最佳浓度.纯化获得N末端携带多聚组氨酸标签的BTV-1 VP2重组蛋白,分子质量约105 ku,Western blotting、ELISA、阻断ELISA结果显示,重组BTV-1 VP2蛋白能与BTV-1型特异性抗体发生特异性结合,且此结合能被BTV-1阻断.本试验结果表明,通过原核表达载体pEASY-Blunt E1表达的BTV-1 VP2重组蛋白具有良好的免疫反应性,为BTV-1 VP2蛋白型特异性表位定位研究奠定了基础.%The study was aimed to test the immunoreactivity of the VP2 protein of bluetongue virus serotype 1 (BTV-1) in vitro.Based on the published BTV-1 L2 gene of Y863 strain, specific cloning PCR primers were designed and synthesized.The L2 gene was amplified through RT-PCR method and then was purified and cloned into the expressing vector pEASY-Blunt E1.The cloned recombinant plasmids were identified.The positive recombinant L2 plasmid was cloned into BL21(DE3) competent cells to express VP2 protein.The acquired purified recombinant BTV-1 VP2 protein was analyzed through the methods of Western blotting, ELISA and blocking ELISA.The results showed that: BTV-1 VP2 protein was expressed as the inclusion bodies in the pEASY-Blunt E1 vector;160 and 200 mmol/L glyoxaline were the best condition to wash down the expressed protein.The molecular weight of this purified recombinant protein with N-terminal His-tag was about 105 ku.Through the results of Western blotting, ELISA and blocking ELISA, it had been proved that this recombinant protein could combine with BTV-1 specific antibody and this combination could be blocked by BTV-1 virus.The study showed that the recombinant BTV-1 VP2 protein, expressed through the prokaryotic expression vector pEASY-Blunt E1, possessed good immunoreactivity and this study had established foundation for locating the serotypic epitopes of the BTV-1 VP2 protein.