抗CD133胞外区骆驼多克隆抗体的制备及鉴定  

Preparation and Characterization of Polyclonal Antibody Against CD133 Derived from Camel

作　　者：翟田甜[1] 马晓玲[1] 木亚沙尔.买买提拉洪 李玲霞[1] 李江伟[1] 

机构地区：[1]新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《中国生物工程杂志》2014年第8期24-28,共5页China Biotechnology

基　　金：国家自然科学基金资助项目(81260333;31370933)

摘　　要：目的:制备高效价、高特异性的抗CD133胞外区新疆双峰驼来源的多克隆抗体,为制备高亲和力的抗CD133纳米抗体做准备。方法:将CD133胞外区基因序列构建到原核表达载体pET28a中,诱导表达及纯化CD133蛋白,免疫新疆双峰驼及新西兰兔。通过酶联免疫吸附实验(ELISA)和Western blot检测多克隆抗体的效价及与CD133特异性结合活性。结果:ELISA测定抗-CD133骆驼源抗体的效价可达到百万以上,通过Western blot检测多克隆抗体可特异性结合CD133蛋白。结论:重组人CD133蛋白可以在骆驼体内激发高滴度抗体反应,为今后构建骆驼免疫单域抗体噬菌体展示文库奠定基础。Objective： The purpose is confirming and acquiring of high titer anti-CD133 antibodies from immunized camel,and providing the basis for preparing anti-CD133 nanobody. Methods： The gene segment coding of CD133 extracellular region were PCR amplified and ligated into pET28 a plasmid and construction of a prokaryotic expression vector. The recombinant CD133 protein was expressed by IPTG induction and purified with Ni-affinity chromatography. A male Bactrian camel and two New Zealand rabbits were immunized with purified rCD133 antigen respectively. The enzyme-linked immunosorbent（ ELISA） and Western blotting were applied to assay the titer of polyclonal antibody and the specific binding to CD133 protein. Results： the ELISA results revealed that the titer of anti-CD133 antibody raised from camel reached about 1∶ 106 after the 5thimmunization,and the titer of anti-CD133 antibody raised from rabbit reached about 1∶ 5 × 105 after the 4thimmunization. The anti-CD133 antisera could bind rCD133 specifically in Western blotting. Conclusion： The high titer anti-CD133 polyclonal antibodies were raised in camel compared with rabbit and it lay a good foundation for the future experiment.

关 键 词：CD133 多克隆抗体 新疆双峰驼 肿瘤干细胞 

分 类 号：Q786[生物学—分子生物学]