水禽核糖体蛋白S12与番鸭细小病毒结构蛋白VP1的体外相互作用研究

Study on in vitro interaction between waterfowl ribosomal protein S12 and Muscovy duck parvovirus structural protein VP1

为了探讨水禽核糖体蛋白S12(ribosomal protein S12,RPS12)与番鸭细小病毒(Muscovy duck parvovirus,MDPV)结构蛋白VP1之间的相互作用关系,试验根据GenBank中已登录的北京鸭和浙东白鹅RPS12基因序列,人工合成重组质粒pUC57-DRPS12和pUC57-GRPS12,双酶切后回收目的片段,分别插到载体pGEX-6P-1中,构建重组质粒pGEX-DRPS12和pGEX-GRPS12。将pGEX-DRPS12、pGEX-GRPS12及含MDPV YL08株VP1基因的重组质粒pET-VP1分别转化至大肠杆菌Rosetta(DE3)感受态细胞中,IPTG诱导表达,利用HRP标记的GST或His标签单克隆抗体,通过Western-blot法鉴定重组蛋白,采用GST pull-down法体外检测水禽RPS12蛋白与VP1蛋白的相互作用情况。结果表明:获得的重组蛋白GST-DRPS12、GST-GRPS12和TRX-VP1分子质量分别为41.2,40.3,98.8 ku,浓度为1.32,1.29,0.81 mg/mL;重组蛋白TRX-VP1能够与MDPV YL08株感染的番鸭血清特异性结合,具有较好的反应原性;重组蛋白TRX-VP1可以和GST-DRPS12、GST-GRPS12在体外相互作用。说明MDPV VP1蛋白与水禽RPS12蛋白存在相互作用关系。

In order to investigate the interaction between waterfowl ribosomal protein S12(RPS12) and Muscovy duck parvovirus structural protein VP1, according to the RPS12 gene sequences of Pekin duck and Zhedong White goose recorded in GenBank, the recombinant plasmids pUC57-DRPS12 and pUC57-GRPS12 were artificially synthesized. After double enzyme digestion, the target fragments were recovered and inserted into the vector pGEX-6 P-1 to construct recombinant plasmids pGEX-DRPS12 and pGEX-GRPS12. The pGEX-DRPS12, pGEX-GRPS12 and recombinant plasmid pET-VP1 containing the VP1 gene of MDPV YL08 strain were transformed into competent E. coli Rosetta(DE3) respectively. The expression was induced by IPTG, and GST or His tag monoclonal antibodies labeled with HRP were used;the recombinant proteins were identified by Western-blot method. GST pull-down method was used to detect in vitro interaction between waterfowl RPS12 protein and VP1 protein. The results showed that the molecular masses of the obtained recombinant proteins GST-DRPS12, GST-GRPS12 and TRX-VP1 were 41.2, 40.3 and 98.8 ku, respectively;the concentrations were 1.32,1.29 and 0.81 mg/mL, respectively. The recombinant protein TRX-VP1 could specifically bind to the serum of Muscovy ducks infected by MDPV and had good antigenicity. The recombinant protein TRX-VP1 could interact with GST-DRPS12 and GST-GRPS12 in vitro. The results suggested that MDPV VP1 protein had an interaction relationship with waterfowl RPS12 protein.