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目的利用大肠杆菌原核表达系统表达肠道病毒71型强神经毒分离株VP1蛋白,为抗体制备和病毒诊断试剂盒的开发奠定基础。方法利用一步法RT-PCR扩增出病毒VP1基因,构建重组原核表达质粒,IPTG诱导后经SDS-PAGE和Western blot验证蛋白表达的特异性。结果重组质粒在1 mmol/L的IPTG 37℃诱导6 h后可诱导表达得到约33 KD的蛋白,且表达的蛋白主要以不溶性的包涵体形式存在。VP1蛋白特异性单抗和His蛋白单抗验证了蛋白表达的特异性。结论获得特异性的EV71病毒河南分离株VP1蛋白,可用于开发病毒诊断试剂盒。
Objective To express the VP1 protein of enterovirus 71 type neurovirulent strain by using prokaryotic expression system of E. coli, which lays the foundation for antibody preparation and development of virus diagnostic kit. Methods The virus VP1 gene was amplified by one-step RT-PCR and the recombinant prokaryotic expression plasmid was constructed. The specificity of protein expression was verified by SDS-PAGE and Western blot after induced by IPTG. Results The recombinant plasmid could induce the expression of about 33 KD protein induced by 1 mmol / L IPTG at 37 ℃ for 6 h, and the expressed protein mainly existed as insoluble inclusion body. The specificity of protein expression was verified by VP1 protein-specific mAb and His-protein monoclonal antibody. Conclusion The specific VP71 protein of EV71 virus isolated from Henan province can be used to develop the virus diagnostic kit.