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当前位置: 首页 > 文章 > 蓝舌病毒VP6蛋白的原核表达及抗体制备 中国兽医科学 2017 (5) 603-609
Position: Home > Articles > Prokaryotic expression and antibody preparation of bluetongue virus VP6 protein Chinese Veterinary Science 2017 (5) 603-609

蓝舌病毒VP6蛋白的原核表达及抗体制备

作  者：
张国芮;独军政;高闪电;田占成;常惠芸
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;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甘肃省动物寄生虫病重点实验室;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农业部畜禽病毒学重点开放实验室国家口蹄疫参考实验室
关键词：
蓝舌病毒;VP6基因;原核表达;多克隆抗体;免疫荧光
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。
译  名：
Prokaryotic expression and antibody preparation of bluetongue virus VP6 protein
作  者：
ZHANG Guo-rui;DU Jun-zheng;GAO Shan-dian;TIAN Zhan-cheng;CHANG Hui-yun;YIN Hong;XUE Hui-wen;College of Veterinary Medicine,Gansu Agricultural University;State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses;
关键词：
bluetongue virus;;VP6 gene;;prokaryotic expression;;polyclonal antibody;;immunofluorescence
摘  要：
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 pProEx-HTb to construct the recombinant plasmid pPro-VP6. The expression plasmid pPro-VP6 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. Immunofluorescence assay revealed that the VP6 proteins were expressed in the whole cytoplasm of BTV-infected BHK21 cells. In conclusion, the rVP6 of BTV1 were expressed in E.coli and its polyclonal antibodies were prepared, which laid a foundation for further studies on the VP6 protein.

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