人SUMO-3基因原核表达及多克隆抗体制备

构建人SUMO-3基因的原核表达载体pET41a(+)-SUMO-3，表达重组GST-SUMO-3融合蛋白，制备人SUMO-3多克隆抗体。试验结果显示，通过PCR方法从重组质粒pEYFP-SUMO-3中克隆到的SUMO-3 N端93个氨基酸的基因序列与NCBI上提供的序列一致，重组质粒pET41a(+)-SUMO-3构建成功；重组pET41a(+)-SUMO-3在E.coli.BL21 (DE3) pLysS中表达GST-SUMO-3融合蛋白，分子量为44.0 kDa，与预期分子量一致；采用亲和层析纯化融合蛋白GST-SUMO-3并免疫家兔，获得人SUMO-3抗体；Western blot 检测显示该抗体可以特异性识别SUMO-3，ELISA检测结果成阳性，抗体效价约为1: 20000。实验结果为进一步研究人SUMO-3及SUMO第二类家族的功能提供了有用工具。

Prokaryotic expression and preparation of polyclonal antibody for human SUMO-3 gene

To construct a prokaryotic expression vector of Human SUMO-3, purify Human SUMO-3 proteins produced by the expression system, and prepare its antiserum. The fragment of human SUMO-3 gene was amplified from pEYPF-SUMO-3 plasmid by PCR. The enzyme digested target fragment was cloned into pET41a(+) clone and expression vector and transfected into E. coli. BL 21 (DE3) plysS, in which SUMO-3 expression was induced by IPTG. After the soluble protein was purified through GST affinity chromatography, processed by identified by SDS-PAGE, a rabbit was immunized with the fusion protein, and the antiserum was obtained. The result of DNA sequence analysis showed that the cloned SUMO-3 gene sequence was completely corresponding to GenBank data. SDS-PAGE and Western blotting showed that the expressed SUMO-3 fusion protein was about 44.0 kDa, mainly existing soluble protein of E. coli. , that could be purified through GST affinity chromatography. The results of ELISA is positive, and Western blotting confirmed that the antiserum reacted specifically to the SUMO-3 protein. A recombinant SUMO-3 protein and the specific polyclonal antibody have been obtained, which provides a basis for establishment of immunoassays of human SUMO-3.