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目的针对碱性成纤维细胞生长因子(b FGF)与成纤维生长因子受体1c(FGFR1Ⅲc)的结合位点,制备鼠抗人b FGF单克隆抗体并研究其生物学活性。方法用重组表达的人b FGF免疫Balb/c小鼠,取小鼠脾脏常规融合制备杂交瘤细胞,通过间接和竞争ELISA筛选出与FGFR1Ⅲc有共同结合位点的抗b FGF单抗。制备小鼠腹水并利用Protein G亲和柱纯化腹水单抗,SDSPAGE鉴定纯化产物;间接ELISA测定单抗亚类、亲和常数,竞争ELISA测定抗体IC50;Western blot和免疫荧光对单抗的生物学活性初步鉴定;CCK-8法检测单抗对肺癌细胞株LL/2的增殖抑制活性。结果筛选出了2株稳定表达b FGF抗体的细胞株Mab E12和Mab D9,竞争ELISA测得其IC50分别为1.564和1.96μg/ml,免疫球蛋白分类两者均为Ig G1,间接ELISA测得Mab E12的亲和常数为5.66×108L/mol;Western blot和免疫荧光表明该抗体能与LL/2细胞内天然合成的b FGF相结合,并对其增殖产生了较明显的抑制作用。结论成功建立了1株能特异性识别b FGF-FGFR1Ⅲc合位点的单抗,能有效的中和b FGF对肺癌细胞株LL/2促增殖作用,为研究肿瘤治疗途径奠定了基础。
OBJECTIVE To prepare monoclonal anti-human bFGF monoclonal antibody for the binding site of basic fibroblast growth factor (b FGF) and fibroblast growth factor receptor 1c (FGFR1Ⅲc) and study its biological activity. Methods Balb / c mice were immunized with recombinant human bFGF. The hybridoma cells were prepared by routine fusion of mouse spleen. The anti-b FGF monoclonal antibody co-binding to FGFR1Ⅲc was screened by indirect and competitive ELISA. The ascites of mice were prepared and the ascites were purified by Protein G affinity column. The purified products were identified by SDSPAGE. The McAbs were subclassified by indirect ELISA, and the affinity constants were determined by ELISA. The IC50 of antibodies was determined by Western blot and immunofluorescence. CCK-8 assay was used to detect the proliferation inhibitory activity of mAb on LL / 2 lung cancer cell line. Results Two Mab E12 and Mab D9 cell lines stably expressing b FGF antibody were screened. IC50 values were 1.564 and 1.96 μg / ml for competitive ELISA, Ig G1 for both immunoglobulins and indirect ELISA Mab E12 affinity constant of 5.66 × 108L / mol; Western blot and immunofluorescence showed that the antibody with LL / 2 cells in the natural synthesis of b FGF binding, and its proliferation had a more significant inhibitory effect. Conclusion A monoclonal antibody capable of specifically recognizing the bFGF-FGFR1Ⅲc locus was successfully established and could effectively inhibit the proliferation of lung cancer cell line LL / 2 induced by bFGF, which lays the foundation for the study of tumor therapeutic pathways.