【摘要】 目的克隆大鼠谷氨酸受体2(GluR2)基因片段,构建重组表达质粒,在原核系统诱导表达,制备多克隆抗体,分析GluR2与caspase3的相互作用。方法从大鼠脑组织提取总RNA,实时聚合酶链反应(RT-PCR)扩增GluR2基因抗原性较强的一段,定向克隆入表达载体pGEX-4T1,构建GST-GluR2融合蛋白表达质粒,转化至大肠杆菌BL21,经IPTG诱导表达GluR2蛋白。用SDS-PAGE方法分离目的蛋白,免疫家兔,制备抗血清。用Western Blot方法鉴定抗体的特异性,用Co-IP实验分析GluR2和caspase3的相互作用。结果SDS-PAGE显示GST-GluR2融合蛋白诱导表达成功;Western Blot显示制备的多克隆抗体具有较高的特异性;Co-IP显示大鼠脑组织中GluR2和caspase3存在相互作用。结论成功获得兔抗鼠GluR2特异性抗血清;脑组织中GluR2和caspase3存在相互作用,为进一步研究神经毒作用导致GluR2下调的分子机制奠定基础。更多还原

【Abstract】 Objective To clone the gene fragment of rat GluR2,construct the recombinant expression vector,express GluR2 fusion protein in prokaryotic system,develop polyclonal antibody against the recombinant protein,and analyze the interaction between GluR2 and caspase3.Methods Total RNA was extracted from brain tissue of rats.RT-PCR was used to amplify the highly antigenic gene fragment of GluR2.The products of amplification were sub-cloned into prokaryotic expression vector pGEX-4T1.GST-GluR2 fusion protein was induced and expressed by IPTG in E.coli BL21 and purified by SDS-PAGE.The purified protein was inoculated into rabbit to develop polyclonal an-tibodies.The specificity of antibodies was evaluated by Western Blot.Co-IP was performed to analyze the interaction between GluR2 and caspase3.Results SDS-PAGE showed that GST-GluR2 fusion protein was expressed successful-ly in E.coli BL21.Western Blot showed high specificity of the polyclonal antibodies.The interaction between GluR2 and caspase3 was evidenced by Co-IP.Conclusion The antibodies against rat GluR2 were developed successfully.The interaction between GluR2 and caspase3 was observed in rat brain tissue.These results can be useful for study of the molecular mechanism of GluR2 down-regulation caused by neurotoxicity.更多还原