蓝舌病毒VP6蛋白的原核表达及抗体制备

发布时间：2018-06-02 06:54

本文选题：蓝舌病毒 + VP基因　；参考：《中国兽医科学》2017年05期

【摘要】：本试验首先利用RT-PCR技术扩增获得了蓝舌病毒1型(BTV1)的VP6基因,然后将其克隆到表达载体pPRoEx-HTb上,构建重组表达质粒pPro-VP6,转化大肠杆菌BL21感受态细胞后用IPTG诱导表达,优化表达条件,纯化重组表达的VP6蛋白,免疫新西兰白兔制备抗VP6蛋白的多克隆抗体,利用Western-blot和细胞免疫荧光试验检测抗体的特异性及VP6蛋白在BTV1感染细胞中的分布。结果显示,重组VP6蛋白在大肠杆菌中以包涵体的形式表达,利用组氨酸标签纯化树脂获得了高纯度的VP6蛋白;制备的抗VP6多克隆抗体不仅可与重组表达的VP6蛋白反应,而且可与BTV1感染细胞中的天然VP6蛋白发生特异性反应;在BTV1感染的BHK21细胞中检测到了VP6蛋白的特异表达,且分布于整个细胞质中。本研究成功表达了BTV重组VP6蛋白并制备了相应的特异抗体,为进一步揭示VP6蛋白的特性和功能奠定了基础。
[Abstract]:In this experiment, the VP6 gene of blue tongue virus type 1 (BTV1) was amplified by RT-PCR technique, and then cloned into the expression vector pPRoEx-HTb. The recombinant expression plasmid pPro-VP6 was constructed. The recombinant plasmid pPro-VP6 was transformed into Escherichia coli BL21 receptive cells, and the expression conditions were optimized by IPTG. The recombinant VP6 protein was purified and the polyclonal antibody against VP6 protein was prepared by immunizing New Zealand white rabbits. The specificity of the antibody and the distribution of VP6 protein in BTV1 infected cells were detected by Western-blot and cellular immunofluorescence assay. The results showed that the recombinant VP6 protein was expressed as inclusion body in E. coli and purified with histidine label resin to obtain high purity VP6 protein, and the polyclonal antibody against VP6 could not only react with the expressed VP6 protein. The specific expression of VP6 protein was detected in BTV1 infected BHK21 cells and distributed in the whole cytoplasm. In this study, the recombinant VP6 protein of BTV was successfully expressed and the corresponding specific antibody was prepared, which laid a foundation for further revealing the characteristics and functions of VP6 protein.
【作者单位】：甘肃农业大学动物医学院;中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心;
【基金】：甘肃省国际科技合作专项(1504WKCA056) 国家自然科学基金项目(31672562)
【分类号】：S852.65

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