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目的制备S100A9重组蛋白及其单克隆抗体(mAb),并对抗体进行特异性鉴定。方法以成人肝cDNA表达文库为模板,构建重组表达质粒pGEX-4T-1-S100A9和pET-32a-S100A9。融合蛋白在大肠杆菌中表达,纯化后以His-S100A9作为免疫原制备鼠mAb。采用ELISA和Western blot法鉴定筛选抗体。挑取杂交瘤细胞株制备腹水并纯化,采用Western blot法和间接免疫荧光染色鉴定mAb的特异性。结果 GST-S100A9和His-S100A9融合蛋白均成功构建表达。共筛选到抗S100A9杂交瘤细胞18株,其中15株在Western blot检测中与重组蛋白呈强阳性反应,3株呈弱阳性反应。2株可识别肝癌组织间隙液中的S100A9天然蛋白。结论成功地制备出多株抗S100A9的mAb。
Objective To prepare S100A9 recombinant protein and its monoclonal antibody (mAb) and to specifically identify the antibody. Methods The recombinant expression plasmids pGEX-4T-1-S100A9 and pET-32a-S100A9 were constructed using adult liver cDNA expression library as a template. The fusion protein was expressed in E. coli. After purification, the murine mAb was prepared using His-S100A9 as an immunogen. Antibodies were screened by ELISA and Western blot. The hybridoma cell lines were selected for preparation of ascites and purified. The specificity of mAb was identified by Western blot and indirect immunofluorescence staining. Results Both GST-S100A9 and His-S100A9 fusion proteins were successfully constructed. A total of 18 anti-S100A9 hybridoma cells were screened, of which 15 showed strong positive reaction with Western blot and 3 weakly positive reaction. Two strains can identify S100A9 natural protein in interstitial fluid of liver cancer. Conclusion A number of mAbs against S100A9 were successfully prepared.