【作者】 马吉春；

【导师】 台桂香；

【作者基本信息】 吉林大学 ， 免疫学， 2007， 硕士

【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。更多还原

【Abstract】 MUC1 is the mucin that is a membrane protein composed of core peptide and carbohydrate side chain expressed on normal epithelia cells and adenocarcinoma cells. Normal MUC1 exists on ductal epithelia as a large heavily glycosylated protein is expressed only on the apical cell surface directed the duct lumen, and it is isolated with the immunological cell. When the tissue becoming cancerate, the construction of MUC1 is up-regulated on tumour cells where it undergoes changes in deglycosylation and distributes on whole tumor cell surface, which make it distinct from normal mucin. Therefore, the tumor-specific epitopes hidden in the normal are exposed, which make it a novel target for active tumor immunodetection and immunotherapy. Different products which are the splicing after the gene transcription encoding the protein are the isotype of MUC1. The MUC1 protein in the peripheral blood is the MUC1/SEC, which lock the inner segment of MUC1/REP and is the full sequence of outer segment. It has been reported that the solvable MUC1 protein in the peripheral blood of many adenocarcinoma patients including breast cancer, lung cancer, pancreatic cancer, colorectal cancer and so on is higher than the normal,and it is associated with malignant extent of tumour. So the detection of the secretory level of MUC1 protein in peripheral blood by diagnosis and monitoring the prognosis for many adenocarcinoma patients are very valuable in clinical application.At present, the conventional methods of MUC1 protein detection in the peripheral blood contains RT-PCR, ELISA, immunocytochemical stain, flow cytometry and so on. RT-PCR is the most sensitive technique for detection, but it has a high false positive rate and the process of operation is tedious. Commercial ELISA is low in detection rate. Both immunocytochemical stain and flow cytometry are poor in sensitivity and the operations are complicated.Our research aim at detecting the level of the MUC1 protein in peripheral blood of many adenocarcinoma patients, and establish a more sensitive indirect sandwich ELISA method. We prepared two polyclonal antibodies that were anti human MUC1 protein and established the method of MUC1 protein detection in adenocarcinoma patient by indirect sandwich ELISA.Methods and Results:1. 459bp fragment of MUC1 was inserted into pGEX-4T-1 expression vector, and successfully constructed recombinant pGEX - MUC1.2. MUC1-GST fusion protein was expressed by Ecoli BL21 transformed by the recombinant, and the pGEX-MUC1/ BL21 was obtained.3. A large pGEX-MUC1/ BL21 cultured in LB medium were broken by sonicate and removed supernatant containing fusion protein. The MUC1-GST protein was purified by Glutathione Sepharose 4B affinity chromatography and further MUC1-GST fusion protein was purified by non-denaturing PAGE.4. A large pMAL- MUC1/DH5αcultured in LB medium were broken by sonicate and removed supernatant containing fusion protein. The MUC1-MBP protein was purified by amylose resin affinity chromatography.5. The rabbit and rats were immunized by MUC1-GST fusion protein and MUC1-MBP fusion protein respectively. We obtained the high potency rabbit anti human MUC1 protein polyclonal antibody and rat anti human MUC1 protein polyclonal antibody. The titers of two antibodies were 320000 and 33250±8220respectively.6. We successfully established the indirect sandwich ELISA method for MUC1 protein detection by sieved the different groups of MUC1 fusion protein and the anti human MUC1 polyclonal antibodies.7. We detected MUC1 protein in peripheral blood serum of 32 breast cancer patients and 20 healthy people using the indirect sandwich ELISA. The result displayed that the positive detection rate was 53.1% in breast cancer group, the rate of the healthy people group was 0.The indirect sandwich ELISA method we established for MUC1 protein detection is convenient, fast, characteristic, sensitive, widespread and high detection. We hope it would become a convention kit in clinical application for early diagnosis and monitoring the prognosis in adenocarcinoma patient.更多还原