肠道病毒71 VP1基因的原核表达及其抗血清的制备

发布时间：2018-02-03 04:13

本文关键词： 肠道病毒 VP 抗血清抗体效价　出处：《免疫学杂志》2013年01期 　论文类型：期刊论文

【摘要】：目的克隆并表达重组肠道病毒71(EV71)VP1基因,进行抗血清的制备并检测抗血清效价。方法利用原核表达系统将PCR获得的VP1片段构建成原核表达载体pET32a(+)-VP1,诱导其表达VP1融合蛋白并利用包涵体纯化方法进行重组蛋白的纯化,将纯化的重组蛋白免疫新西兰兔制备VP1抗血清,ELISA检测抗体效价。同时构建真核表达载体,利用其在真核细胞中的瞬时表达检测抗血清的特异性。结果通过克隆获得VP1原核表达载体,IPTG诱导VP1蛋白表达并纯化,SDS-PAGE结果显示重组蛋白表达且纯化浓度较高,将重组蛋白乳化后免疫新西兰兔3次,取血检测抗血清的效价,ELISA结果显示抗体效价为1∶64 000,利用所构建真核表达载体pcDNA3.1(+)-VP1表达VP1对抗血清进行检测,Western blot结果表明抗血清可较好的与VP1特异性结合。结论制备具有免疫原性的VP1蛋白及其效价较高的抗血清,为进一步研究抗EV71诊断方法、血清学诊断试剂盒及抗病毒疫苗的研制奠定基础。
[Abstract]:Objective to clone and express the recombinant enterovirus EV71 VP1 gene. Methods the prokaryotic expression vector pET32a (pET-VP1) was constructed from the VP1 fragment obtained from PCR by using prokaryotic expression system. The recombinant protein was induced to express VP1 fusion protein and purified by inclusion body method. The purified recombinant protein was immunized with New Zealand rabbit to prepare VP1 antiserum. At the same time, the eukaryotic expression vector was constructed, and the specificity of antiserum was detected by transient expression in eukaryotic cells. Results the prokaryotic expression vector of VP1 was obtained by cloning. IPTG induced the expression of VP1 protein and purified SDS-PAGE results showed that the recombinant protein was expressed in high concentration. The recombinant protein was emulsified and immunized with New Zealand rabbits for 3 times. The titer of antiserum was detected by Elisa. The titer of antibody was 1: 64 000. Using the constructed eukaryotic expression vector pcDNA3.1 (VP1) to express VP1 antiserum for detection. The results of Western blot showed that the antiserum could bind to VP1 specifically. Conclusion the immunogenicity of VP1 protein and the antiserum with high titer were prepared. It lays a foundation for the further study of anti-EV71 diagnostic method, serological diagnostic kit and anti-virus vaccine.
【作者单位】：东北农业大学生命科学学院;山东省农业科学院奶牛研究中心;济南军区军犬训练大队;
【基金】：国家自然科学基金(31272586) 国家转基因重大专项(2009ZX08007-006B,2011ZX08007-002,2011ZX08008-004) 山东省青年自然科学基金项目(ZR2010ZR029) 山东省自然科学基金(ZR2010CM012) 济南市高校院所主创新计划(201004027)
【分类号】：R373
【正文快照】： 肠道病毒71型(Enterovirus 71,EV71)是导致手足口病(hand,foot and mouth disease,HFMD)以及心肌炎、肺水肿、无菌性脑膜炎等多种并发症的主要病原体之一,多发于5岁以下儿童,以1～3岁最为常见[1]。EV71属于小RNA病毒科肠道病毒属,病毒颗粒为二十面立体对称的球型结构,直径为2

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