鼠抗人FL单克隆抗体的制备及其生物学功能研究

发布时间：2018-05-27 13:15

本文选题：FL + 杂交瘤　；参考：《苏州大学》2010年硕士论文

【摘要】： 目的研制鼠抗人Flt3-ligand(FL)单克隆抗体(mAb)并对其生物学功能进行研究。方法采用高表达膜FL的基因工程细胞株L929/FL细胞免疫BALB/c小鼠,利用B淋巴细胞杂交瘤技术进行细胞融合,免疫荧光标记和流式细胞术筛选阳性株,采用快速定性试纸法鉴定单抗亚类、间接免疫荧光法检测单抗对肿瘤细胞株的识别谱,然后研究所获得的抗人FL单克隆抗体及FL蛋白对肿瘤细胞生长的影响。结果得到稳定分泌鼠抗人FL单克隆抗体的杂交瘤细胞三株,命名为5H5、5D1、3E5,快速定性试纸法鉴定5H5、5D1属小鼠IgM亚类及κ型,3E5属小鼠IgG1亚类及κ型;经体内诱生腹水法制备抗体,小鼠腹水形成的阳性率约为80%,腹水的收获量平均为2 ml/只小鼠;经免疫亲和层析法纯化,腹水中抗体蛋白的得率为1.0 mg/ml;用流式细胞术分析细胞膜荧光标记百分率和荧光强度,结果显示,FL能不同程度地在某些肿瘤细胞株上表达。将FL蛋白和3E5加入到U937、675321、THP-1细胞的培养体系中,经细胞计数分析,3E5能够阻断FL蛋白对U937、675321、THP-1细胞生长的促进作用。结论本研究获取三株分泌鼠抗人FL单克隆抗体的杂交瘤细胞,且3E5为一株阻断型单克隆抗体,这为研究FL及Flt3功能奠定了物质基础。
[Abstract]:Purpose Mouse anti-human Flt 3 ligand FLL) monoclonal antibody was prepared and its biological function was studied. Method BALB/c mice were immunized with genetically engineered cell line (L929/FL) with high expression membrane FL. The BALB/c mice were fused with B lymphocyte hybridoma technique. The positive strains were screened by immunofluorescence labeling and flow cytometry. The monoclonal subclasses were identified by rapid qualitative test paper method. Indirect immunofluorescence assay was used to detect the recognition spectrum of the monoclonal antibody against human FL and the effect of FL protein on the growth of tumor cells. Result Three hybridoma cells stably secreting monoclonal antibody against human FL were obtained, named as 5H5H5O5D1O3E5. Rapid qualitative test paper was used to identify the subclasses of IgM and IgG1 and 魏 type of 5H5D5D1D1E5 and 5H5D5E5, respectively, and to prepare the antibody by the method of inducing ascites in vivo. The positive rate of ascites formation in mice was about 80%, the average yield of ascites was 2 ml/ in mice, the yield of antibody protein in ascites was 1.0 mg / ml by immunoaffinity chromatography, and the fluorescence labeling percentage and fluorescence intensity of cell membrane were analyzed by flow cytometry. The results showed that FL could be expressed in some tumor cell lines to some extent. FL-protein and 3E5 were added to the culture system of U937O675321 THP-1 cells. The cell count analysis showed that FL-protein could block the growth of U937C675321 cells. Conclusion In this study, three hybridoma cells secreting mouse anti-human FL monoclonal antibody were obtained, and 3E5 was a blocking monoclonal antibody, which laid a material foundation for studying the function of FL and Flt3.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【共引文献】