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目的构建人TIM-3融合蛋白原核表达载体,获取蛋白质抗原,制备抗人TIM-3单克隆抗体。方法通过特异性引物从人外周血单个核细胞中扩增出hTIM-3cDNA,细胞膜外基因克隆到原核表达载体pQE100S中,转化到E.coliBL21(DE3),表达并纯化带6个组氨酸的融合蛋白,免疫BALB/c小鼠,应用杂交瘤技术经克隆筛选获得抗人TIM-3单抗的杂交瘤细胞株,以间接酶链反应吸附测定(ELISA)和WesternBlot等方法进行单克隆抗体的鉴定。结果人TIM-3cDNA经DNA测序得到证实,成功构建了pQE100S-hTIM-3原核表达载体,表达并纯化了大量蛋白质抗原,获得了1株稳定分泌抗人TIM-3单抗的杂交瘤细胞株(28H12),WesternBlot分析证明hTIM-3蛋白在转染的CHO细胞中高表达。结论成功构建了人TIM-3融合蛋白原核表达载体,表达并纯化了6×His-hTIM-3融合蛋白,所获单抗能特异识别人TIM-3蛋白,为进一步研究人TIM-3及其配体的功能提供了条件。
Objective To construct a prokaryotic expression vector of human TIM-3 fusion protein and obtain protein antigens to prepare anti-human TIM-3 monoclonal antibody. Methods hTIM-3 cDNA was amplified from human peripheral blood mononuclear cells by specific primers. The extracellular gene was cloned into prokaryotic expression vector pQE100S and transformed into E.coli BL21 (DE3). The recombinant plasmid was expressed and purified with 6 histidine The fusion protein was used to immunize BALB / c mice. The hybridoma cells were screened by hybridoma technique to obtain the monoclonal antibody against human TIM-3 monoclonal antibody. The McAbs were detected by indirect enzyme-linked immunosorbent assay (ELISA) and Western Blot Identification. Results The human TIM-3 cDNA was confirmed by DNA sequencing. The prokaryotic expression vector pQE100S-hTIM-3 was successfully constructed, a large number of protein antigens were expressed and purified, and a hybridoma cell line stably secreting anti-human TIM-3 mAb 28H12). Western blot analysis demonstrated that hTIM-3 protein is highly expressed in transfected CHO cells. Conclusion The prokaryotic expression vector of human TIM-3 fusion protein was successfully constructed, and the 6 × His-hTIM-3 fusion protein was expressed and purified. The obtained monoclonal antibody can specifically recognize human TIM-3 protein. To further study the effect of human TIM-3 and its Ligand function provided the conditions.