【摘要】 旨在探讨番鸭细小病毒(MDPV)非结构蛋白NS1与水禽真核翻译延伸因子1A1(eEF1A1)之间的互作关系。根据GenBank中已发表的北京鸭和浙东白鹅eEF1A1基因序列,经大肠杆菌偏爱密码子优化,人工合成的质粒分别命名为pUC57-DeEF1A1和pUC57-GeEF1A1;对含有MDPV YL08株NS1基因的重组质粒pUC57-NS1、pUC57-DeEF1A1和pUC57-GeEF1A1进行BamHⅠ和XhoⅠ双酶切,回收目的片段后分别插入到原核表达载体pGEX-6p-1和pET-32a中,构建了重组质粒pGEX-DeEF1A1、pET-GeEF1A1、pET-NS1和pGEX-NS1;重组质粒分别转化至大肠杆菌Rosetta(DE3),经IPTG诱导,获得了重组蛋白GST-DeEF1A1、TRX-GeEF1A1、TRX-NS1和GST-NS1的表达;采用HRP标记的GST单克隆抗体鉴定GST-DeEF1A1、HRP标记的His单克隆抗体鉴定TRX-GeEF1A1;采用HRP标记的His单克隆抗体鉴定TRX-NS1、HRP标记的GST单克隆抗体鉴定GST-NS1,采用MDPV感染番鸭血清鉴定TRX-NS1和GST-NS1的抗原性;采用GST pull-down试验验证GST-DeEF1A1与TRX-NS1、GST-NS1与TRX-GeEF1A1的互作。结果表明,获得的重组蛋白GST-DeEF1A1、TRX-GeEF1A1、TRX-NS1和GST-NS1的分子量分别为76.9,67.9,89.5,98.6 ku; GST-DeEF1A1和GST-NS1可与HRP标记的GST单克隆抗体特异性结合,TRX-GeEF1A1和TRX-NS1可与HRP标记的His单克隆抗体特异性结合;GST-NS1和TRX-NS1可与MDPV感染番鸭血清特异性结合,抗原性良好;GST pull-down试验中,GST-DeEF1A1可与TRX-NS1互作,GST-NS1不与TRX-GeEF1A1互作。本研究表明,MDPV NS1蛋白可以和北京鸭eEF1A1在体外相互作用而不能和浙东白鹅eEF1A1在体外相互作用,互作结果差异性可能与二者的102-106 aa, 108 aa的氨基酸残基有关。更多还原

【Abstract】 This study was conducted to investigate the interaction relation between Muscovy duck parvovirus non-structural protein NS1 and waterfowl eukaryotic translation elongation factor 1 A1(eEF1 A1). According to the published eEF1 A1 gene sequence of Pekin duck and Zhedong white goose in GenBank, the artificially synthesized plasmids were named pUC57-DeEF1 A1 and pUC57-GeEF1 A1 respectively after optimization of the preferred codons of E. coli;The recombinant plasmids pUC57-NS1,pUC57-DeEF1 A1 and pUC57-GeEF1 A1 containing the NS1 gene of MDPV YL08 strain were digested with BamHⅠand XhoⅠ,and the target fragments were recovered and inserted into the prokaryotic expression vectors pGEX-6 p-1 and pET-32 a to construct recombinant plasmids pGEX-DeEF1 A1,pET-GeEF1 A1,pET-NS1 and pGEX-NS1,respectively; The recombinant plasmids were transformed into E. coli Rosetta(DE3),and induced by IPTG,the expression of recombinant proteins GST-DeEF1 A1,TRX-GeEF1 A1,TRX-NS1 and GST-NS1 were obtained; The HRP-labeled GST monoclonal antibody was used to identify GST-DeEF1 A1 and the HRP-labeled His monoclonal antibody was used to identify TRX-GeEF1 A1;The HRP-labeled His monoclonal antibody was used to identify TRX-NS1 and the HRP-labeled GST monoclonal antibody was used toidentify GST-NS1,and the MDPV infected Muscovy duck serum was used to identify the antigenicity of TRX-NS1 and GST-NS1;GST pull-down test was used to verify the interaction between GST-DeEF1 A1 and TRX-NS1,GST-NS1 and TRX-GeEF1 A1. The results showed that the molecular weights of GST-DeEF1 A1,TRX-GeEF1 A1,TRX-NS1 and GST-NS1 were 76.9,67.9,89.5,98.6 ku, respectively; GST-DeEF1 A1 and GST-NS1 could specifically bind to HRP-labeled GST monoclonal antibody, while TRX-GeEF1 A1 and TRX-NS1 could specifically bind to HRP-labeled His monoclonal antibody; GST-NS1 and TRX-NS1 could specifically bind to MDPV infected duck serum with good antigenicity; In GST pull-down test, GST-DeEF1 A1 could interact with TRX-NS1,but GST-NS1 could not interact with TRX-GeEF1 A1. This results showed that MDPV NS1 protein could interact with Pekin duck eEF1 A1 in vitro but not with Zhedong white goose eEF1 A1 in vitro and the interaction difference might be related to the 102-106 aa and 108 aa amino acid residues.更多还原