肝癌细胞膜蛋白单克隆抗体的筛选与制备

发布时间：2018-01-19 23:35

本文关键词： 肝癌细胞膜抗原单克隆抗体杂交瘤技术抗原纯化　出处：《重庆医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:提取肝癌细胞膜蛋白作为抗原,利用杂交瘤抗体技术筛选并制备针对肝癌细胞膜蛋白的单克隆抗体。方法:分别利用研磨方法(第一部分)、膜蛋白提取试剂盒(第二部分)、低渗法(第三部分)提取肝癌细胞HepG2细胞膜蛋白作为抗原,超速离心法纯化抗原。纯化后Bradford法进行蛋白定量。用提取的抗原每次25ug-50ug剂量免疫Balb/c小鼠;以聚乙二醇法融合免疫小鼠脾细胞与SP20骨髓瘤细胞,用HAT、HT选择培养基筛选杂交瘤;以提取的肝癌细胞膜蛋白作为包被抗原,用ELISA方法筛选能分泌抗肝癌细胞膜蛋白单克隆抗体的阳性杂交瘤细胞克隆;扩大培养所获得的单克隆细胞,制备单克隆抗体,检测所获得单克隆抗体与肝癌细胞膜蛋白的反应性。结果:研磨方法提取膜蛋白,免疫四次后,ELISA检测免疫小鼠血清抗体效价达1:105,尾静脉冲击免疫后进行细胞融合,融合四周后,共有10株阳性克隆细胞,扩大培养后共有5株克隆生长,但ELISA检测均为阴性;膜蛋白提取试剂盒提取膜蛋白,50ug剂量免疫小鼠共四次后,ELISA检测免疫小鼠血清抗体效价均为阴性,用真空干燥法去除有机溶剂后再次免疫小鼠,小鼠血清抗体效价仍为阴性,免疫失败;低渗法提取膜蛋白,免疫小鼠五次后,ELISA检测免疫小鼠血清抗体效价达1:106,细胞融合后四周,共有22株阳性克隆细胞,扩大培养后共有15株克隆生长,但多次ELISA检测细胞培养上清均为阴性。结论:用研磨方法及低渗法提取肝癌细胞膜蛋白作为抗原,制备针对肝癌细胞膜蛋白的单克隆抗体存在一定缺陷。这两种方法所提取的膜蛋白纯度不高、获得的膜蛋白量小,细胞膜蛋白抗原成分多,用所提取的膜蛋白制备单克隆抗体相对比较困难。本实验目的是针对其中各种单一抗原制备单克隆抗体,故研磨方法中以每次25ug剂量免疫小鼠,剂量显然不足,故在试剂盒方法及低渗法提取膜蛋白时增加免疫剂量至50ug。用所提取的细胞膜蛋白免疫小鼠,血清中产生的是多克隆抗体,针对单一抗原所产生的血清效价低于针对细胞膜蛋白抗原的血清效价,故应尽量待血清效价达1:106后再进行细胞融合。试剂盒提取膜蛋白纯度及定量优于研磨方法,但试剂盒中含有的有机溶剂可能影响了膜蛋白的抗原性,真空干燥法可能不能完全清除有机溶剂,从而影响免疫小鼠产生抗体的效果。
[Abstract]:Aim: to select and prepare monoclonal antibodies against hepatoma cell membrane proteins by using hybridoma antibody technique. Methods: HepG2 cell membrane proteins were extracted by grinding method (part I), membrane protein extraction kit (part II) and hypoosmotic method (part III). The antigen was purified by ultracentrifugation. The protein was quantified by Bradford method. Balb/c mice were immunized with the extracted antigen at a dose of 25ug-50ug each time. Spleen cells of immunized mice and SP20 myeloma cells were fused with polyethylene glycol method. The ELISA method was used to screen the positive hybridoma cell clones which secreted monoclonal antibodies against hepatoma cell membrane protein. The monoclonal antibody was prepared and the reactivity of the monoclonal antibody to hepatoma cell membrane protein was detected. Results: the membrane protein was extracted by grinding method. The titer of serum antibody of immunized mice was 1: 105 by Elisa after four times immunization. There were 10 positive clones and 5 clones grew after expanded culture, but ELISA was negative. The serum antibody titers of mice immunized with 50ug of membrane protein extraction kit were negative after four times of immunization with Elisa. After removing the organic solvent by vacuum drying method, the mice were immunized again, the antibody titers of the mice were still negative and the immunity failed. The membrane protein was extracted by hypotonic method. The titer of serum antibody of immunized mice was 1: 106 by Elisa after five times. Four weeks after cell fusion, there were 22 positive clone cells. A total of 15 clones grew after expanded culture, but the supernatant of cell culture detected by ELISA was negative. Conclusion: there are some defects in the preparation of monoclonal antibody against hepatoma cell membrane protein by grinding method and hypotonic method. The purity of membrane protein extracted by these two methods is not high. The amount of membrane protein obtained is small, the membrane protein antigen components are many, it is relatively difficult to prepare monoclonal antibody with the extracted membrane protein. The purpose of this experiment is to prepare monoclonal antibody against a variety of single antigens. Therefore, the grinding method to each dose of 25ug immunization mice, the dose is obviously insufficient. Therefore, when the membrane protein was extracted by Kit and hypoosmotic method, the immune dose was increased to 50ug.To immunize mice with the extracted membrane protein, the polyclonal antibody was produced in the serum. The titer of serum produced against single antigen was lower than that against membrane protein antigen. The purity and quantification of membrane protein extracted by the kit was better than the grinding method, but the organic solvent contained in the kit might affect the antigenicity of the membrane protein. Vacuum drying may not completely remove organic solvents, thus affecting the antibody production in immunized mice.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【引证文献】