重组MUC1融合蛋白的制备及检测MUC1双抗体夹心ELISA方法的建立

发布时间：2018-05-14 17:38

本文选题：MUC1 + 重组MUC1融合蛋白　；参考：《吉林大学》2007年硕士论文

【摘要】： MUC1粘蛋白因其在多种腺癌包括乳腺癌、卵巢癌、肺癌等细胞上的高度异常表达使其成为肿瘤诊断和治疗的有效靶点。研究发现,腺癌患者尤其乳腺癌患者外周血中MUC1的水平较正常人高。其水平升高可以作为肿瘤辅助诊断指标。但目前因方法各异,在临床上尚未形成一种成熟的敏感特异的技术手段,而国外研发商业化生产的双抗体夹心ELISA法(CASA test)对腺癌的检出阳性率较低。本研究为检测肿瘤患者外周血中MUC1蛋白水平,建立一种更加敏感的双抗体夹心ELISA技术。首先构建pGEX- MUC1和pMAL-MUC1两种表达载体,转化大肠杆菌,通过IPTG诱导MUC1-GST及MUC1-MBP融合蛋白在大肠杆菌中的表达;经Glutathione Sepharose 4B亲和层析和非变性聚丙烯酰胺凝胶电泳纯化MUC1-GST融合蛋白,通过Amylose Resin亲和层析纯化MUC1-MBP融合蛋白,经Western blot鉴定蛋白的正确性;然后,将纯化的MUC1-GST和MUC1-MBP融合蛋白分别免疫家兔和大鼠,制备两种抗人MUC1多克隆抗体。最后,通过不同组合筛选合适的一级抗体、二级抗体及标准品,建立MUC1蛋白的双抗体夹心ELISA检测方法,采用双抗体夹心ELISA方法检测乳腺癌患者血清中MUC1蛋白含量。本研究所建立的双抗体夹心ELISA检测血清中MUC1的方法,具有简便、快捷、敏感性强及检出率高等优点,可望发展成为临床上检测腺癌的一种常规试剂盒。
[Abstract]:Due to its highly abnormal expression in many adenocarcinoma cells, including breast, ovarian, lung cancer, MUC1 mucin has become an effective target for tumor diagnosis and treatment. The level of MUC1 in peripheral blood of adenocarcinoma patients, especially breast cancer patients, was higher than that of normal controls. The elevated level can be used as an auxiliary diagnostic index of tumor. However, due to different methods, a mature sensitive and specific technique has not yet been developed in clinic, and the positive rate of detection of adenocarcinoma by developed and commercialized double antibody sandwich ELISA test is low. In order to detect the level of MUC1 protein in peripheral blood of tumor patients, a more sensitive sandwich ELISA technique with double antibodies was established. Firstly, two expression vectors, pGEX- MUC1 and pMAL-MUC1, were constructed and transformed into Escherichia coli, and MUC1-GST and MUC1-MBP fusion proteins were induced to express in E. coli by IPTG. MUC1-GST fusion protein was purified by Glutathione Sepharose 4B affinity chromatography and non-denaturing polyacrylamide gel electrophoresis. The MUC1-MBP fusion protein was purified by Amylose Resin affinity chromatography and identified by Western blot. Then the purified MUC1-GST and MUC1-MBP fusion proteins were immunized with rabbit and rat respectively to prepare two kinds of anti-human MUC1 polyclonal antibodies. Finally, by screening suitable primary, secondary and standard antibodies in different combinations, a double antibody sandwich ELISA method for detection of MUC1 protein was established, and the content of MUC1 protein in serum of breast cancer patients was detected by double antibody sandwich ELISA method. The method of double antibody sandwich ELISA for detection of MUC1 in serum is simple, rapid, sensitive and high detection rate. It is expected to develop into a routine kit for the detection of adenocarcinoma in clinic.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【相似文献】