抗CD47胞外区骆驼多克隆抗体的制备及鉴定  

Preparation and Identification of Polyclonal Antibody Against CD47 Derived from Camel

作　　者：贾二坷 冯亚宁[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：《生物技术通报》2015年第8期200-205,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31370933)

摘　　要：验证人CD47蛋白能否在新疆双峰驼中产生高滴度抗体;为制备高亲和力的抗CD47纳米抗体提供实验依据。构建CD47胞外区原核表达载体,诱导表达及纯化CD47蛋白,免疫新疆双峰驼。通过酶联免疫吸附试验(ELISA)和Western blot分别检测骆驼多克隆抗体的效价及与CD47蛋白特异性结合活性。结果显示,成功构建CD47胞外区原核表达载体,获得纯度大于90%的CD47蛋白,表达纯化的CD47重组蛋白可与抗CD47单抗B6H12.2特异结合;ELISA测定CD47重组蛋白免疫骆驼7次后,抗CD47多克隆抗血清的效价至少达到1∶200 000,免疫印迹检测表明抗CD47骆驼抗血清与CD47重组蛋白、Jurkat和Raji细胞表面表达的CD47均能特异结合。重组人CD47蛋白可以在骆驼体内激发高滴度抗体反应。This work aims to verify whether or not recombinant human CD47 would induce high antibody response in Xinjiang Camelus bactrianus, and provide the experimental evidences for preparing anti-CD47 nanobody of high affinity. The gene segment of CD47 extracellular region was amplified by PCR and ligated into pET30a plasmid, and a prokaryotic expression vector was constructed. The recombinant CD47 protein （rCD4） was expressed by IPTG induction and purified with Ni-affinity chromatography. A male C. bactrian camel was immunized with purified rCD47 antigen. The enzyme-linked immunosorbent （ELISA） and Western blotting were applied to assay the titer of polyclonal antibody and the specific binding to CD47 protein. The purified rCD47 of high purity （90%） from prokaryotic expression vector of CD47 extracellular region bound specifically with B6H12.2 of anti-CD47. The ELISA results revealed that the titer of polyclonal anti-CD47 antibody in camel at least reached 1：200000 after the 7th immunization. The Western blotting results demonstrated that camel antiserum of resisting CD47 specifically bound with rCD47, and CD47 on membrane of Jurkat and Raji cells. In conclusion, the anti-CD47 polyclonal antibodies of high titer were raised in camel and it lay a promising foundation for the future experiment.

关 键 词：CD47 多克隆抗体 新疆双峰驼 纳米抗体 

分 类 号：Q813.2[生物学—生物工程]