关键词: CD47, 多克隆抗体, 新疆双峰驼, 纳米抗体

Abstract: 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.

Key words: CCD47, polyclonal antibody, Bactrian camel, nanobody