抗GD2单链抗体-IL-2融合蛋白基因的构建及表达

将已经突变了稀有密码子的人IL-2（Ala125）基因，与GD2单链抗体基因通过over-lap-PCR法，构建成ScFv-IL-2融合基因，并在基因的C端引入his tag序列。ScFv-IL-2基因经测序正确后连接到表达载体pSE380上，然后导入大肠杆菌BL21中表达。表达蛋白为包涵体，表达量占总蛋白的30％以上，经SDS-PAGE及Western blot鉴定表达蛋白的分子量为43kD，与预期的相符。包涵体粗提后，经Ni^2＋亲和层析柱及Sephacryl S-200HR柱分离复性，纯度可达90％以上。ELISA试验结果证明该融合蛋白保存了与相应抗原的结合活性和IL-2的生物活性。

Construction and Expression of an Anti- GD2,ScFv-IL-2 Fusion Protein Gene

By combining interleukin2(IL-2) with a tumor specific antibody,immunotherapy of tumors may become more effective in the future.Anti-GD2 single chain antibody directed to the extracellular domain of GD2 disialoganglioside can result in an antitumor response in some pateins with tumors expressing GD2.In this study,the fusion protein consisting of GD2 single chain antibody(ScFv) and IL-2(Ala125) was constructed.Anti-GD2 ScFv and IL-2 genes were obtained by PCR,then the ScFv-IL-2 gene was constructed by over lap PCR.The gene was inserted into the pMD18-T easy vector.Genes from pMD18-T-vector were inserted into expression vector pSE380.Recombinant expression vector was identified by restriction enzyme-cutting and then was transformed into BL21.SDS-PAGE and Western blot analysis confirmed that the transformed E.Coli BL21 could express ScFv-IL-2 fusion-proteins and the molecular weight is 43 kDa.The fusion protein was purified by affinity chromatograph and Sephacryl S-200HR then was identified through ELISA.The results show that the fusion protein retains the activities of both antigen binding and IL-2.