蓝舌病毒VP6蛋白的原核表达及抗体制备

Prokaryotic expression and antibody preparation of bluetongue virus VP6 protein

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。

In this study, the VP6 gene of bluetongue virus serotype 1 （BTV1） was amplified by reverse transcription-polymerase chain reaction（RT-PCR） and inserted into prokaryotic expression vector pPro- Ex-HTb to construct the recombinant plasmid pPro-VP6. The expression plasmid pPro-YP6 was transformed into E. coli BL21 competent cells and induced with IPTG. The expression parameters were optimized and the recombinant VP6 proteins（rVP6） were purified by His-Tag Purification Resin. New Zealand white rabbits were immunized with rVP6 to prepare the polyclonal antibodies. The specificity of the antibodies was evaluated by Western-blot and immunofluorescence assay. The results showed that the rVP6 were expressed as inclusion bodies in E. coli and purified successfully. Western-blot showed that the polyclonal antibodies were able to react with the rVP6 as well as the natural VP6 proteins in BTV1 infected cells. Im- munofluorescence assay revealed that the YP6 proteins were expressed in the whole cytoplasm of BTV-in- fected BHK21 cells. In conclusion, the rVP6 of BTV1 were expressed in E. coli and its polyclonal antibod-ies were prepared, which laid a foundation for further studies on the VP6 protein