肠道病毒71型vp1基因的扩增 克隆 表达及蛋白分析-amplification, cloning, expression and protein analysis of vp1 gene of enterovirus 71.docxVIP

32KD 的融合表达蛋白，与预期分子量相符。通过包涵体纯化得到 VP1 高纯度蛋白，以纯化蛋白包被反应板检测 EV71 阳性、COX A16 阳性病人的血清标本，EV71 阳性病 人 OD 值均值 2.385±0.628，COX A16 阳性病人 OD 值均值 1.231±0.428，正常对照 组 OD 值均值 0.384±0.128。EV71 VP1 蛋白检测敏感性和特异性分别为 83.3%和 85%。结论：成功构建了EV71 VP1基因高效原核表达载体pET28a-EV71 VP1，并获得蛋白的大 量表达；通过对感染病人血清的ELISA初步分析，得出EV71 VP1敏感性和特异性分别 为83.3%和85%，为后续的疫苗和检测试剂盒研究奠定基础。关键词：肠道病毒 71 型；VP1；克隆；原核表达；酶联免疫吸附测定Amplification, cloning, expression and protein analysisof Enterovirus 71 VP1 gene Applicant: Yunfeng Liu Advisor: Zhenwen ZhouABSTRACTObjective:Enterovirus 71 (EV71) was belongs to a small RNA virus family which causes hand-foot-mouth disease (HFMD) and nervous system diseases such as Aseptic Meningitis, Brainstem Encephalitis and Poliomyelitis paralysis. In recent years, HFMD incidence rate significantly increased, and the distributed trends turn to throughout the year from seasonal. EV71 VP1 protein was the main viral neutralization factor, which directly determines the antigenicity of the virus. Therefore, the study of the EV71 VP1 protein antigenicity and other biological characteristics has important clinical significance.The EV71 VP1 gene was amplified, cloned, and expressed in prokaryotic expression system. Sequence analysis, bioinformatics analysis, purification of VP1 were done. Also, recombinant VP1 was used to test patients immunoreactive by ELISA, which laid the foundation for the diagnosis and vaccine research.Method:A pair of specific primers was designed according to GenBank EV71 sequence (EU703813.1), viral RNA was extrated from childrens throat swab specimens. EV71 VP1 gene was RT-PCR amplified by using viral RNA as a template. The PCR product was double enzyme digestion by BamH I and Xho I, the product was linked with the corresponding double enzyme digestion pET28a vector, then transformed into E.coli DH5a. Kanamycin (Kana) positive colonies plasmid was extracted and identified by double enzyme digestion. The gene was subcloned into expression pET28a vector, subsequently expressed in E.coli B