大鼠激活转录因子3单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibody against rat activation transcription factor 3

目的制备特异性识别大鼠激活转录因子3(ATF3)的单克隆抗体(mAb)。方法根据软件预测的结果合成偶联有匙孔绒血蓝蛋白(key holeli mpet hemocyanin,KLH)的大鼠ATF3第168-181位氨基酸多肽。随后免疫BALB/c小鼠,运用杂交瘤技术制备抗大鼠ATF3的mAb。采用快速定性试纸法鉴定该单克隆抗体的亚型和亚类;通过ELISA、Western blot和免疫组织化学法鉴定单克隆抗体的特性。结果获得一株可稳定分泌抗大鼠ATF3 mAb的杂交瘤细胞,其分泌的ATF3单抗的亚型是IgG1,轻链为κ链。Western blot结果显示,该单抗能够特异性识别大鼠细胞内表达的ATF3蛋白。免疫组织化学检测结果表明,在Thy-1肾炎(Thy-1N)大鼠肾细胞胞质及细胞核内均有ATF3表达,但细胞核内的表达量较为显著。结论成功制备了一株抗大鼠ATF3的mAb,为进一步研究ATF3的生物学功能提供了可用的实验材料。

Objective To prepare a hybridoma secreting stable monoclonal antibody against rat activation transcription factor 3（ATF3）.Methods Peptides（168-181 amino acids of rat ATF3 protein） were synthesized according to predicted antigenic peptides sequence and coupled to keyhole limpet hemocyanin（peptide-KLH）.Then mAb against peptide-KLH was prepared by hybridoma technique.A commercial mouse mAb isotyping kit was used to identify the isotype of this mAb.Meanwhile,ELISA,Western blot and IHC were used to determine whether the mAb could bind rat ATF3 protein.Results One hybridoma secreting stable mAb against rat ATF3 was produced.Immunoglobulin subclass determination identified this mAb to be the IgG1/κ subtype.Western blot result revealed that the mAb could specifically recognize rat ATF3 protein in glomerular mesangial cells（GMC）.Immunohistochemical staining showed that the mAb could react with ATF3 expressed both in the cytoplam and the nuclei of GMC in rats with Thy-1 nephritis while the expression of ATF3 in the nuclei was more significant.Conclusion A mouse mAb against rat ATF3 protein was prepared successfully,which provides a useful tool for studying ATF3 biological function in the future