人SUMO-2基因原核表达、纯化及多克隆抗体制备

Prokaryotic expression,purification and preparation of polyclonal antibody for human SUMO-2 gene

目的:构建人SUMO-2基因的原核表达载体,纯化融合蛋白GST-SUMO2-SUMO2并以其为抗原免疫家兔,制备人SUMO-2多克隆抗体。方法:用PCR的方法得到人SUMO-2基因并克隆至pET41a(+)原核表达载体中,转化大肠杆菌BL21(DE3)plysS诱导融合蛋白GST-SUMO2-SUMO2表达;所获得的可溶性蛋白经亲和层析纯化、SDS-PAGE电泳鉴定后,免疫家兔制备抗血清,分别采用ELISA、Western blot检测抗体效价和特异性。结果:测序证实重组质粒pET41a(+)-SU-MO2-SUMO2构建成功;SDS-PAGE结果证实获得Mr为52000的GST-SUMO2-SUMO2融合蛋白且为可溶性蛋白;经过GST亲和层析有效纯化;以该融合蛋白免疫家兔制备得到的抗血清经Western blot检测证实能与目的蛋白发生特异性结合,ELISA检测为阳性。结论:获得了人SUMO-2蛋白及特异性多克隆抗体,对进一步研究人SUMO-2及SUMO第二类家族的功能提供了有用工具。

AIM： To construct a prokaryotic expression vector of human SUMO-2, purify GST-SUMO2-SUMO2 fusion protein produced by the expression system, and prepare its antiserum. METHODS： The human SUMO-2 gene was amplified by PCR. The target fragment digested by the enzyme was cloned into a pET41a（ ＋ ） expression vector and then transfected into E. coli. BL21 （DE3） pLysS, in which GST-SUMO2-SUMO2 fusion protein was induced by IPTG. After the soluble protein was purified by GST affinity chromatography and by identified by SDS-PAGE, the rabbits were immunized with the fusion protein and the antiserum was obtained. RESULTS： DNA sequence analysis showed the cloned SUMO-2 gene sequence was completely corresponding to GenBank data. SDS-PAGE and Western blot showed that the GST-SUMO2-SUMO2 fusion protein was about 52 kDa, which was mainly the soluble protein of E. coil and could be purified by GST affinity chromatography. The result of ELISA was positive and Western blot confirmed the antiserum reacted specifically to SUMO-2 protein. CONCLUSION： SUMO-2 protein and its specific polyclonal antibody have been prepared, which provides a basis for the establishment of immunoassays of human SUMO-2.