人TIM-3的克隆表达及其单克隆抗体的制备

Cloning, expression of human TIM-3 and preparation of its monoclonal antibody against it

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 the prokaryotic expression vector of human T-cell immunoglobulin mucin 3 （TIM-3） and obtain recombinant human TIM-3 protein to prepare mouse anti-human TIM-3 monoclonal antibody for further study of immunological function of human TIM-3 and its role in the pathogenesis of autoimmune diseases. Methods The human TIM-3 cDNA was cloned from human peripheral blood mononuclear cells （PBMC）. The extracellular domain of human TIM-3 was inserted into prokaryotic expression vector pQE100S and transformed into E. coli BL21（DE3）. The 6xHis-tagged TIM-3 protein was expressed in E. coil BALB/c mice were immunized with recombinant protein, hybridoma cells were screened by cell fusion and subcloning approach. The monoclonal antibody was identified by ELISA and Western blot. Results The human TIM-3 protein was expressed at high level in E. coli and purified under denaturing conditions. The monoclonal antibody 28H12 was obtained with Ig subclass of IgG2a. Western blot analysis confirmed that the mAb 28H12 specifically recognized the TIM-3 expressed on transfected CHO cells. Conclusion The extracellular gene of TIM-3 has been cloned into prokaryotic expression vector pQE100S and expressed in E.coli. The mAb 28H12 can bind specifically to human TIM-3.