人SUMO-1基因原核表达、纯化及多克隆抗体制备与鉴定

Prokaryotic expression,purification,identification,and polyclonal antibody preparation of human SUMO-1 gene

目的构建人SUMO-1基因的原核表达载体,诱导表达重组蛋白,制备兔抗人SUMO-1抗血清。方法从质粒pcDNA-HA-SUMO1-GG中用PCR方法克隆到编码人SUMO-1N端97个氨基酸的基因片段,构建了SUMO-1原核表达载体pET41a(+)-SUMO1,转化大肠杆菌BL21(DE3)pLysS,IPTG诱导蛋白表达并利用GST亲和层析柱进行纯化,以纯化后的融合蛋白GST-SUMO1为抗原免疫家兔,获得抗血清。Western blotting、ELISA法鉴定获得的抗血清。结果成功构建原核表达载体,纯化到融合蛋白GST-SUMO1,用纯化的融合蛋白免疫家兔制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论成功获得了人SUMO-1多克隆抗体,为进一步研究人SUMO-1蛋白的功能奠定了基础。

Objective To construct a prokaryotic plasmid expressing the recombinant proteins of human SUMO-1 and prepare the polyclonal antibodies of rabbit anti-human SUMO-1.Methods The fragment of SUMO-1 gene encoding 97 amino acids in the N terminus was amplified from pcDNA-HA-SUMO-1-GG plasmid by PCR.The fragment was then inserted into the prokaryotic expression vector to construct the recombinant plasmid pET41a(+)-SUMO1,and expressed in E.coli.BL21(DE3)pLysS.After induction with IPTG,the fusion protein was obtaine...