【摘要】 目的:研究参与铁硫蛋白形成的关键分子BolA同系物3(BolA3)在细胞中的功能,通过重组表达BolA3蛋白,进而免疫制备并鉴定针对该分子的抗体。方法:从人肺癌细胞A549细胞中提取RNA,RT-PCR扩增出BolA3基因,构建BolA3基因重组原核表达载体pQE30-BolA3,使用IPTG诱导蛋白表达,Western blotting法鉴定表达中带His标签的重组BolA3蛋白,使用镍(Ni)柱纯化该蛋白。程序免疫大耳白兔,酶联免疫吸附试验(ELISA)方法测定兔血清中抗体滴度。Western blotting法检测制备的抗体与真核表达的BolA3蛋白结合的特异性,并利用已制备的BolA3蛋白抗体检测多种细胞中BolA3内源性表达。结果:克隆的BolA3基因序列与理论序列相一致,原核表达并纯化了BolA3重组蛋白,程序免疫大耳白兔后,获得了高滴度、高特异性的抗体血清。使用所得纯化的BolA3蛋白抗体检测到多种细胞内源性表达BolA3。结论:成功制备针对BolA3的多克隆抗体,为进一步深入研究BolA3蛋白的生物学功能奠定了基础。更多还原

【Abstract】 Objective:To prepare and identify the recombinant polyclone antibody against BolA homolog 3(BolA3),a key factor of iron-sulfur proteins formation,and to study its function in cells.Methods:BolA3was cloned from the cDNA which was amplified from the human lung cancer A549 cells by RT-PCR,then subcloned into the prokaryotic expression vector pQE30.The expression of His-tagged BolA3 was induced by IPTG and purified by His Trap affinity column.The recombinant protein was used to immunize rabbit.The serum antibody titerin rabbits was detected by enzyme-linked immunosorbent assay(ELISA).The specificity of antibody combined with BolA3 protein in several human cell lines were identified by western blotting.Results: The recombinant prokaryotic expression vector pQE30-BolA3 was successfully constructed.The high titer and high specificity of antibodies were obtained in rabbit serum.Western blotting showed that the purified BolA3 protein was expressed in several human cell lines.Conclusion:The polyclone antibody against BolA3 was successfully prepared,which laid the foundation for the further research of BolA3’s biological function.更多还原