摘要
目的:制备鼠抗人程序性细胞死亡分子10(programm ed cell death 10,PDCD10)的单克隆抗体,探讨PDCD10蛋白的结构和功能。方法:利用重组PDCD10蛋白为免疫原,免疫BALB/C小鼠,取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞Sp2/0进行常规融合,通过间接ELISA的筛选和有限稀释克隆化,获得鼠抗PDCD10蛋白单克隆抗体的杂交瘤细胞株,通过ELISA,W estern b lot和免疫荧光实验等方法对其特性进行鉴定。结果:成功地建立了3株稳定分泌抗PDCD10蛋白的单克隆抗体杂交瘤细胞株,分别命名为5G1,4F7和3H5。3株单克隆抗体的免疫球蛋白亚类分别为IgG1(4F7和5G1)和IgG2b(3H5)。ELISA检测的单克隆抗体腹水效价可达1∶107。3株单克隆抗体与重组PDCD10蛋白有较强的特异性反应,而与大肠杆菌细胞裂解液以及谷胱苷肽-S转移酶(glutath ioneS-transferase,GST)没有交叉反应。同时,5G1单克隆抗体也能特异性地结合真核细胞内源性和超表达的PDCD10蛋白,免疫荧光竞争结合实验以及W estern b lot的结果证明3株PDCD10的单克隆抗体能够识别不同的抗原表位,内源性以及超表达的PDCD10蛋白主要定位在细胞核。结论:获得了效价高、特异性好的PDCD10蛋白的单克隆抗体,为PDCD10的生物学功能研究奠定了基础。
Objective： To obtain monoclonal antibodies against programmed cell death 10 （PDCD10） for further study of the structure and function of PDCD10 protein. Methods： Balb/c mice were immunized with recombinant PDCD10, hybridoma cell lines secreting monoclonal antibodies against PDCD10 were screened by regular cell fusion and subcloning approach. The specificities of these monoclonal antibodies were determined by ELISA, Western blotting and Immunofluorescecence assay. Results： Three hybridoma cell lines （5G1, 4F7 and 3H5 ） stable in secreting specific monoclonal antibodies were successfully obtained. Subclass of IgG belonged to IgG1 （4F7 and 5G1 ） and IgG2b（ 3H5 ）, respectively. The ascite titers of these monoclonal antibodies reached 1：10^7. They could specifically bind to recombinant PDCD10 and endogenous and overexpressed PDCD10 proteins proved by ELISA and Western blotting. They failed to react with E. coli lysates and glutathione S-transferase （GST）. In addition, these three monoclonal antibodies could recognize different epitopes of PDCD10 proteins assessed by immune fluorescence competitive binding assay. Both endogenous and overexpressed PDCD10 protein mainly located in the nucleus. Conclusion： Monoclonal antibodies against PDCD10 with high titers and specificity have been successfully prepared, which has laid the foundation for further study of PDCD10 protein.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2006年第6期586-591,共6页
Journal of Peking University:Health Sciences
基金
国家自然科学基金青年基金(30300308)资助~~
