Preparation Of Recombinant MUC1 FusionProtein And Establishment Of MUC1 ProteinDetection By Indirect Sandwich ELISA

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.