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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。
OBJECTIVE: To clone the GluR2 gene fragment of rat and construct a recombinant expression plasmid. The recombinant plasmid was induced in prokaryotic system to produce polyclonal antibody. The interaction between GluR2 and caspase3 was analyzed. Methods Total RNA was extracted from rat brain tissue and amplified by real-time polymerase chain reaction (RT-PCR). The GluR2 gene was cloned into the expression vector pGEX-4T1 and the GST-GluR2 fusion protein expression plasmid was constructed. To E. coli BL21, induced by IPTG GluR2 protein expression. The target protein was separated by SDS-PAGE and immunized rabbits to prepare antiserum. The specificity of the antibody was determined by Western Blot and the interaction between GluR2 and caspase3 was analyzed by Co-IP assay. Results SDS-PAGE showed that the expression of GST-GluR2 fusion protein was successful. Western Blot showed that the polyclonal antibody was highly specific. Co-IP showed the interaction of GluR2 and caspase3 in rat brain. Conclusions The rabbit anti-mouse GluR2-specific antiserum was successfully obtained. GluR2 and caspase3 interacted in brain tissue, which laid the foundation for the further study on the molecular mechanism of neurotoxicity resulting in the down-regulation of GluR2.