C-myc单克隆抗体的制备及鉴定

发布时间：2018-04-03 12:49

本文选题：c-myc　切入点：免疫　出处：《辽宁师范大学》2011年硕士论文

【摘要】：原癌基因c-myc是myc家族的重要成员之一,是髓细胞性白血病病毒的癌基因v-myc的细胞同类物,定位于染色体8q24.12-8q24.13,其编码产物c-myc蛋白的分子量为62kDa。c-myc基因的功能甚多,它既是一种可易位基因,又是一种受多种物质调节、可使细胞无限增殖、获永生化功能、促进细胞分裂的可调节基因,在很多肿瘤中都高表达。在正常细胞中的c-myc原癌基因一旦被激活,转为癌基因,活化转录和翻译,c-myc mRNA和c-myc蛋白的表达便异常增高,使细胞脱离正常生长的调节限制而具有高度增生潜能,向恶性表型转化,抑制细胞分化,促进细胞恶变,最后导致肿瘤的发生。鉴于c-myc的过表达与肿瘤发生呈高度正相关,c-myc抗血清可以应用于肿瘤的诊断,本研究通过制备c-myc单克隆抗体,为肿瘤诊断试剂盒的研发提供基础实验数据。用原核表达及纯化的c-myc蛋白抗原免疫3只健康雌性BALB/c小鼠,3次免疫后取小鼠脾细胞与SP2/0骨髓瘤细胞以10:1比例进行细胞融合,再经HAT选择培养基筛选、间接ELISA方法检测、有限稀释法克隆,最终获得2株能够稳定分泌抗c-myc特异性单克隆抗体的杂交瘤细胞株,并分别命名为SHH-1H11和SHH-2A9。抗体亚型鉴定表明,SHH-1H11、SHH-2A9分别为IgG1亚类和IgM。经BCA Protein Assay Reagent Kit检测显示,杂交瘤细胞SHH-1H11上清和腹水的抗体浓度分别为0.47mg/ml和2.51mg/ml,SHH-2A9的上清和腹水的抗体浓度分别是0.49mg/ml和2.45mg/ml。ELISA检测结果显示,这两株杂交瘤细胞培养上清效价分别为1:10240、1:5120;小鼠腹水效价分别为1:64000、1:32000,c-myc单克隆抗体只与c-myc蛋白反应,与其他蛋白没有交叉反应,这表明获得的单克隆抗体具有较高的特异性和敏感性。本研究为肿瘤诊断试剂盒的研发提供了良好的技术基础。
[Abstract]:Proto-oncogene c-myc is one of the important members of the myc family, and is the cell congener of v-myc, the oncogene of myeloid leukemia virus, which is located on chromosome 8q24.12-8q24.13. The molecular weight of c-myc protein encoded by the proto-oncogene is the function of 62kDa.c-myc gene.It is not only a translocative gene, but also a regulated gene which is regulated by a variety of substances. It can make cells proliferate infinitely, gain immortalized function and promote cell division. It is highly expressed in many tumors.Once the proto-oncogene of c-myc in normal cells is activated, the expression of c-myc mRNA and c-myc protein is increased abnormally, which makes the cells have high proliferative potential without the regulation of normal growth.Transformation to malignant phenotype, inhibition of cell differentiation, promotion of cell malignancy, and eventually tumorigenesis.In view of the highly positive correlation between overexpression of c-myc and carcinogenesis, c-myc antiserum can be used in the diagnosis of tumor. In this study, we prepared monoclonal antibodies against c-myc to provide basic experimental data for the development of tumor diagnostic kit.Three healthy female BALB/c mice were immunized with prokaryotic expression and purified c-myc protein antigen. The spleen cells were fused with SP2/0 myeloma cells at 10:1 ratio after three times immunization. Then the cells were selected by HAT selective medium and detected by indirect ELISA method.Two hybridoma cell lines, named SHH-1H11 and SHH-2A9, were obtained, which could secrete monoclonal antibody against c-myc stably.The identification of antibody subtypes showed that SHH-1H11H11 SHH-2A9 was IgG1 subclass and IgM subgroup respectively.BCA Protein Assay Reagent Kit showed that the antibody concentrations of SHH-1H11 supernatant and ascites were 0.47mg/ml and 2.51mg / ml SHH-2A9, respectively. The results of 0.49mg/ml and 2.45mg/ml.ELISA showed that the antibody concentration of SHH-1H11 supernatant and ascitic fluid were 0.49mg/ml and 2.45mg/ml.ELISA, respectively.The supernatant titers of the two hybridomas were 1: 10240 and 1: 5120, respectively, and the mice ascites titers were 1: 64000 and 1: 32000.The monoclonal antibodies against c-myc reacted only with c-myc protein and did not cross with other proteins, which indicated that the obtained monoclonal antibodies had high specificity and sensitivity.This study provides a good technical basis for the development of tumor diagnostic kit.
【学位授予单位】：辽宁师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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