蓝舌病1型病毒VP2蛋白的原核表达及免疫反应性分析

发布时间：2018-02-01 16:57

本文关键词： 蓝舌病 VP蛋白表达免疫反应性　出处：《中国畜牧兽医》2017年07期 　论文类型：期刊论文

【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-1Y863毒株L2基因序列设计合成特异性BTV-1PCR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-1VP2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200mmol/L咪唑是洗脱BTV-1VP2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-1VP2重组蛋白,分子质量约105ku,Western blotting、ELISA、阻断ELISA结果显示,重组BTV-1VP2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-1VP2重组蛋白具有良好的免疫反应性,为BTV-1VP2蛋白型特异性表位定位研究奠定了基础。
[Abstract]:The aim of this study was to study the bluetongue virus serotype 1 of bluetongue type 1 virus. The expression product of BTV-1)VP2 protein was immunoreactive in vitro. Specific BTV-1PCR primers were designed and synthesized according to the published L2 gene sequence of BTV-1Y863 strain. The L2 gene was amplified by RT-PCR, and the purified L2 gene was cloned into pEASY-Blunt E1 expression vector to identify the recombinant plasmid. The positive recombinant plasmid L2 was cloned into Escherichia coli BL21DDE3) and expressed in the competent cells. The purified BTV-1 VP2 recombinant protein was analyzed by Western blotting and ELISA analysis. BTV-1VP2 protein was expressed in the form of inclusion body on pEASY-Blunt E1 vector, and Ni-NTA affinity chromatography was used. 160 and 200 mmol / L imidazole was the best concentration for elution of BTV-1VP2 protein expression product. BTV-1VP2 recombinant protein with polyhistidine label was purified. The molecular weight of EISAs was about 105 Ku. Western blotting showed that EISAs were blocked by ELISA. The recombinant BTV-1VP2 protein can specifically bind to BTV-1 specific antibody, and this binding can be blocked by BTV-1. The recombinant BTV-1VP2 protein expressed by prokaryotic expression vector pEASY-Blunt E1 has good immunoreactivity. The results laid a foundation for the study of BTV-1VP2 type specific epitope localization.
【作者单位】：云南省热带亚热带动物病毒病重点实验室;
【基金】：国家自然科学基金(31260612) 公益性行业(农业)科研专项(201303035)
【分类号】：S852.65
【正文快照】： 蓝舌病是由蓝舌病病毒(bluetongue virus,BTV)引起的通过库蠓等媒介昆虫传播的反刍动物的虫媒病毒病[1]。BTV主要引起绵羊发病和死亡,牛和山羊常为隐性感染,蓝舌病的发生和分布与媒介昆虫的分布有密切关系,主要暴发于库蠓大量活动的夏秋季节,特别多见于低洼潮湿多水区域,该病

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