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-1 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.
出处
《东南大学学报（医学版）》
CAS
2011年第4期533-537,共5页
Journal of Southeast University(Medical Science Edition)
基金
国家自然科学基金资助项目(30871108)
关键词
大肠癌
CD133抗原
分子克隆
表达
纯化
鉴定
colorectal cancer
CD133 antigen
molecular cloning
expression
purification
identification
