结肠癌组织中CD133抗原的原核表达、纯化和初步鉴定

摘要

目的:制备CD133特异性抗原用于筛选CD133单克隆抗体库,筛选产物能够与肿瘤表面的CD133抗原结合以达到靶向治疗的目的.方法:用PCR扩增CD133两段胞外区,分别将扩增的两段片段与PGEX4T-1(6*HIS)质粒结合,将连接产物转入BL21 大肠杆菌中培养,IPTG诱导表达,镍柱纯化.通过ELISA等技术对纯化后的重组蛋白进行鉴定.结果:已成功表达出CD133两段胞外区,ELISA鉴定表明,纯化后的两段CD133胞外区抗原可特异性地结合CD133抗体.结论:筛选出的两段CD133胞外区抗原可用于CD133特异性抗体的筛选.%Objective : To establish monoclonal antibodies that can specially recognize the tumor surface CD133-antigen. Methods : The two large extracellular sequences of human CD133-antigen were amplified by PCR, cloned into a PGEX4T-I vector having a N-terminal-HIS-tag. The inserted fragment was sequenced and then transformed into BL21( DE3 ) E. coli strain. The constructed plasmids were induced by IPTG to express the recombinant protein and then were purified by using Nickel-NTA beads. Results: It was proven that extracellular region of CD133 antigen could specifically bind to commercial CD133 antibody , which was confirmed by ELISA. Conclusion: The extracellular region of CD133 antigens can be used to screen CD133 antibody library at the next step.