抗PSMD10单克隆抗体的制备和鉴定

Preparation and Identification of Monoclonal Antibodies Ggainst PSMD10

目的制备抗PSMD10的单克隆抗体(McAb),并对其特异性进行鉴定。方法用柱层析纯化的重组PSMD10蛋白免疫BALB/c小鼠,采用杂交瘤技术制备McAb,用ELISA和有限稀释法筛选出分泌高滴度McAb的杂交瘤细胞株,用Protein-G亲和层析纯化接种杂交瘤细胞的小鼠腹水,测定其免疫球蛋白亚类及其效价,Western-blot分析其特异性。通过免疫组织化学染色法检测PSMD10在肝细胞肝癌组织中的表达部位。结果筛选出2株能稳定分泌抗PSMD10单克隆抗体的杂交瘤细胞株,单抗均为IgG2,Western-blot分析显示,2株单抗都与重组的PSMD10发生特异性结合。免疫组织化学染色分析显示,PSMD10表达在肝细胞肝癌的细胞浆内。结论制备的抗PSMD10杂交瘤细胞株能分泌高滴度和高特异性的单克隆抗体,可望用于进一步研究肝细胞肝癌的发生发展、诊断和治疗。

Objective To prepare monoelonal antibody （mAb） against the recombinant PSMD10 （proteasome 26S subunit,non-ATPase,10）, identify its characteristics and investigate the localization of PSMD10 in human hepatoeellular carcinoma （HCC） by immunostaining. Method BALB/e mice were immunized with recombinant PSMD10. Hybridomas were made using PEG4000 as the fusing agent, and isolated by limiting dilution. The mAb was purified by Protein-G affinity chromatography. The isotype, titer and affinity of the mAb were analyzed. Formalin-fixed paraffin-embedded tissues from 12 HCCs were immunostained to investigate the localization of PSMD10 in the carcinoma cells. Result Two particular mAbs named B006 and B009 were obtained. Both mAb are IgG2. The affinity constants （Kaff） of the B006 and B009 are 1.03×10^-9 M^-1 and 2.15×10^-9 M^-1, respectively. Western-blot analysis showed that all of the two mAbs eoule strongly and specifically reacted with the recombinant PSMD10. The PSMD10 is expressed in cytoplasm of the human hepatoeellular carcinoma. Conclusion Two hybridomas eeU lines secreting high titer of mAbs against the recombinant PSMD10 with high speeitleity are established. These monoelonal antibodies are useful for the further research on the hepatocellular eareinoma.