作 者：
赵冬敏;韩凯凯;刘青涛;黄欣梅;杨婧;刘宇卓;章丽娇;田宇杰;王梦瑶;李银
单 位：
江苏省农业科学院兽医研究所/国家兽用生物制品工程技术研究中心/农业农村部兽用生物制品工程技术重点实验室
关键词：
坦布苏病毒;鸭胚成纤维细胞;热休克蛋白70;受体;免疫共沉淀;病毒辅覆蛋白结合分析(VOPBA)
摘 要：
【目的】拓展对坦布苏病毒受体的认知,为研究其在鸭体内的感染机制打下基础。【方法】取9日龄SPF鸭胚制备鸭胚成纤维细胞(DEF),待细胞长成单层后提取DEF膜蛋白,利用免疫共沉淀试验筛选出DEF膜蛋白中可与坦布苏病毒结合的蛋白,经病毒辅覆蛋白结合分析(VOPBA)验证后通过LC-MSMS质谱分析进行鉴定;同时人工合成鸭热休克蛋白70基因(HSP70)序列的开放阅读框(1905 bp),构建重组质粒pET32a-HSP70并转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达,以重组蛋白免疫小鼠制备抗鸭HSP70血清,与DEF细胞共孵育后接种坦布苏病毒,并采用实时荧光定量PCR检测抗血清对病毒感染的抑制作用。【结果】DEF膜蛋白中分子量约70 kD的蛋白可与坦布苏病毒结合,经LC-MSMS质谱分析和序列比对可确定该蛋白即为鸭热休克蛋白70(HSP70)。含重组质粒pET32aHSP70的BL21(DE3)感受态细胞经IPTG诱导4 h后,SDS-PAGE检测发现在分子量约85 kD处出现目的蛋白条带,获得的重组鸭HSP70蛋白主要以包涵体形式进行表达,且能与His标签抗体发生特异性反应。以重组鸭HSP70蛋白免疫小鼠获得的抗鸭HSP70血清可封闭DEF细胞上的HSP70,而竞争性抑制坦布苏病毒感染。【结论】HSP70是坦布苏病毒感染DEF细胞的受体,可作为新的靶点用于抗坦布苏病毒药物设计。
译 名：
Identification of Tembusu virus receptor protein in the duck embryo fibroblasts
作 者：
ZHAO Dong-min;HAN Kai-kai;LIU Qing-tao;HUANG Xin-mei;YANG Jing;LIU Yu-zhuo;ZHANG Li-jiao;TIAN Yu-jie;WANG Meng-yao;LI Yin;Institute of Veterinary Science,Jiangsu Academy of Agricultural Sciences/National Center for Engineering Research of Veterinary Bio-products/Key Laboratory of Veterinary Biologicals Engineering and Technology,Ministry of Agriculture and Rural Affairs;
单 位：
ZHAO Dong-min%HAN Kai-kai%LIU Qing-tao%HUANG Xin-mei%YANG Jing%LIU Yu-zhuo%ZHANG Li-jiao%TIAN Yu-jie%WANG Meng-yao%LI Yin%Institute of Veterinary Science,Jiangsu Academy of Agricultural Sciences/National Center for Engineering Research of Veterinary Bio-products/Key Laboratory of Veterinary Biologicals Engineering and Technology,Ministry of Agriculture and Rural Affairs
关键词：
Tembusu virus;;duck embryo fibroblasts;;heat shock protein 70(HSP70);;receptor;;co-immunoprecipitation;;virus overlay protein binding assay(VOPBA)
摘 要：
【Objective】The aim of the present study was to expand the understanding of the Tembusu virus receptor and lay the foundation for studying its pathogenesis in ducks.【Method】Duck embryo fibroblasts(DEF)were prepared from 9-day-old SPF duck embryo. Membrane protein was extracted from monolayer of DEF. Then proteins that could bind with Tembusu virus was screened by co-immunoprecipitation from DEF membrane proteins. Virus overlay protein binding assay(VOPBA)was used to validate the binding protein. The target protein band was analyzed by LC-MSMS mass spectrometry. The open reading frame(1905 bp)of duck heat shock protein 70 gene(HSP70)was synthesized artificially and the recombinant plasmid p ET32a-HSP70 was constructed. Then pET32a-HSP70 was transformed into Escherichia coli BL21(DE3)and the competent cell was induced by IPTG. In order to prepare anti duck HSP70 serum,mice were immunized by recombinant duck heat shock protein. Duck embryo fibroblasts were pre-incubated with anti-serum followed by Tembusu virus infection. Real-time fluorescence quantitative PCR was employed to determine the inhibition of anti-serum on infection of the virus.【Result】An approximately 70 kD protein in DEF membrane protein bound to Tembusu virus. LCMSMS mass spectrometry and sequence alignment demonstrated that the 70 kD protein was duck heat shock protein 70(HSP70). The recombinant plasmid pET32a-HSP70 containing BL21(DE3)competent cell was induced by IPTG for 4 h.The analysis of SDS-PAGE showed that the target protein band with molecular weight approximately 85 kD was observed.The recombinant duck HSP70 protein was expressed in inclusion bodies,and reacted specifically with antibody against His tag. Mouse anti duck HSP70 serum was prepared by immunization of recombinant duck HSP70. The infection of Tembusu virus was competitively inhibited by anti duck HSP70 serum which blocked the HSP70 in DEF cell.【Conclusion】HSP70 is identified as receptor of Tembusu virus infected DEF cell. It can be a new target for designing antiviral drug against Tembusu virus.
