人CIDE-3蛋白的表达、纯化及多克隆抗体的制备

Prokaryotic expression, purification and polyclonal antibody preparation of human CIDE-3 Protein

目的:制备人诱导细胞死亡的DFF45样效应因子-3(CIDE-3)蛋白兔抗人多克隆抗体并进行鉴定。方法:将原核表达载体pET28a(+)/CIDE-3转化E.coli BL21(DE3),IPTG诱导表达CIDE-3蛋白,经Ni-NTA Agarose纯化后免疫新西兰白兔,获得人CIDE-3蛋白兔抗血清,并通过免疫印迹(Western blot)、酶联免疫吸附试验(ELISA)及免疫组织化学法对抗体进行鉴定。结果:成功地表达、纯化了人CIDE-3蛋白,与弗氏佐剂乳化后,免疫新西兰白兔,获得高效价的人CIDE-3蛋白兔抗血清,ELISA结果证实该抗体具有较高的亲和性,Western blot及免疫组织化学染色显示证实该抗体能够与人CIDE-3蛋白特异性结合,是一种细胞质蛋白。结论:获得了效价高、特异性较强的人CIDE-3蛋白兔抗血清,为进一步研究人CIDE-3奠定了实验基础。

Objective： To prepare rabbit anti - human cell death - inducing DFF45 - like effector - 3 （ CIDE - 3 ） polyclonal antibody. Methods： The prokaryotic expression vector pET28a（ ＋ ）/CIDE -3 was transformed into host strain E. coli BL21 （ DE3 ） and induced by IPTG. After being purified with Ni - NTA Agarose and emulsified with Freund＇s adjuvant, the human CIDE -3 protein was used as antigen to immunize rabbit. The rabbit anti -human CIDE - 3 antibody was analyzed by Western blot, enzyme - linked immunosorbent assay （ELiSA） and immunohistochemistry staining. Results： Human CIDE- 3 protein was expressed and purified successfully. ELISA analysis showed this antibody had high affinity. The results of Western blot and immuhistochemistry staining showed this antibody could specifically recognize CIDE - 3 protein and located in cytoplasm. Conclusion： Rabbit anti - human CIDE - 3 polyclonal antibody with high affinity and specificity was prepared successfully. Those results will help us to study the function of human CIDE - 3 in future.