摘要
目的:构建人肠道病毒71型(human enterovirus 71,EV71)VP1-VP4重组融合蛋白表达体系.方法:构建EV71 VP1-VP4重组融合蛋白原核表达载体转化大肠杆菌DH5α,诱导表达融合蛋白VP1-VP4,SDS-PAGE和Western blot法进行鉴定.用融和蛋白包被ELISA板检测41例已知血清.结果:重组片段VP1-VP4测序结果与设计目的片段序列相符,重组载体构建成功;SDS-PAGE显示融合蛋白约42.8kDa,与预期值一致;Westernblot提示该融合蛋白可以和EV71 VP1、VP4抗体特异性结合.融合蛋白包被ELISA板能准确检测出41例血清中16例EV71阳性血清,与CA16无交叉反应.结论:表达的EV71 VP1-VP4融合蛋白具有良好的抗原性,可作为EV71感染检测抗原,为EV71诊断试剂和疫苗的研究奠定实验基础.
AIM： To construct a prokaryotic vector express- ing human enterovirus 71 （EV71） VP1-VP4 fu- sion antigen. METHODS： A prokaryotic expression plasmid carrying the VP1-VP4 fusion gene was con- structed and transformed into E. coli DH5a. VP1-VP4 fusion protein was induced to express with IPTG. SDS-PAGE and Western blot were performed to detect VP1-VP4 fusion protein. Pu- rified VP1-VP4 fusion protein was coated onto ELISA plates to detect 41 serum samples for screening EV71 positive serum samples. RESULTS： The sequence of recombinant VP1- VP4 fragment was the same as the expectedsequence, indicating that the recombinant vec- tor was successfully constructed. SDS-PAGE showed that the fusion protein had a molecular weight of 42.8 kDa. Western blot showed that fusion protein can be specifically recognized by VP1 antibody and VP4 antibody. Fusion protein coated onto ELISA plates could accurately detect 16 EV71 positive serum samples from 41 serum samples without cross-reactivity with coxsacki- evirus16 （CA16）. CONCLUSION： The VP1-VP4 fusion protein has good antigenicity and can be used as a diagnos- tic antigen to detect EV71 infection. Our results provide a experimental basis for development of EV71 diagnostic kits.
出处
《世界华人消化杂志》
CAS
北大核心
2012年第34期3366-3369,共4页
World Chinese Journal of Digestology
