Purification And Analysis Of Two New Tumor Associated Antigens

Objective: Malignant tumor is one of the diseases that threatens the health of the people severely and has high mortality rate. The traditional cancer treatment method, such as operation, radiotherapy and chemotherapy, has limited success, due to the reason that we don't know the mechanisms of oncogenesis and immune escape of malignant tumor yet. In the past few decades, with the development of molecular biology,it showed that the discover of the tumor associated antigen(TAA) is one of the break through to solve the problems. This is not only important to the essential study of tumor, but also is the hope of early immunodiagnosis and biotherapy to the cancer. With the development and application of serological identification of antigen by recombinant cDNA expression libraries (SEREX) and proteomics gradually, a great number of tumor associated antigens were detected and identified, so the study of tumor has been promoted enormously.Tumor antigen SC (SCAg), as a group of TAA discovered newly, which is recognized by the monoclonal antibody that is prepared by injecting with cell of human colon specimen into the BALB/c mouse, express high in tumor cells of epithelium tissue such as pancreas, intestinum crassum, small intestine and not or little in normal tissue. This is not only the ideal antigen to development new tumor vaccine, but also is important to the study of the etiology, pathology and immunology of the tumor. In this study, the tumor associated antigen SCAg3A and SCAg6 recognized by monoclonal antibodies (MAbs) SC3A and SC6, were isolated and purified with immunoprecipitation, and characteristics of the two proteins were analyzed. It will provide the new antigen for targeted therapy and will be used in the further investigation to cancer, moreover, it will be made as the new tumor DNA vaccine.Methods: The hybridoma cells were generated at our laboratory previously and the ascitic MAbs were prepared by our lab. The tumor cell lines were presented byThe Academy of Military Medical Sciences and Immunoprecipitation kit was produced by Pierce Company.Hybridoma cells cryopreserved in liquid nitrogen were thawed. The cells were cultured and passaged in the routine methods. The antibody was purified by caprylic acid and coupled with the Immunoprecipitation Kit. We identified the cultured cancer cell lines of colon cancer LS-180, SW1116, Cola205, breast cancer cell line SK, lung cancer cell line A549 with immunocytochemistry and choosed the cell line LS-180 which was positive recognized by the MAbs against SCAg. The cell was collected and cleaned with PBS. By centrifugation and dialysis, the crude extract was prepared with the colon cell line LS-180. The target antigens were separated and purified by immunoprecipitation. Finally, the obtained protein antigens were identified through SDS-PAGE, Western blot and Dot blot.Results: The results of immunocytochemistry staining demonstrated that SCAg3A and SCAg6 were expressed in colon cancer cell line LS-180, which located at the cellular membranes and cytoplasm mainly, and they expressed in breast cancer cell line SK lightly, not in other cancer cell lines. Both purified SCAg3A and SCAg6 were showed one electrophoresis strip in SDS-PAGE, and the molecular weight of them were estimated to be approximately 60.1 kD and 59.0 kD respectively. The outcomes of Western blot of SCAg3A and SCAg6 showed that strong signals appeared when the blot was probed with the corresponding MAb. Similar results were seen in the Dot blot and no cross reaction was observed between SCAg3A and SCAg6 in addition.Conclusion: In this study, the tumor associated antigen SCAg3A and SCAg6 were isolated and purified by immunoprecipitation successfully. It appeared one strip in SDS-PAGE which was not interacted by the albumin. The molecular weight of SCAg3A was about 60.1 kD and SCAg6 was about 59.0 kD respectively. No cross reaction among SCAg3A and SCAg6 was aslo found. This results proved that it is feasible that we isolated the high purified antigen SCAg3A and SCAg6 by the cultured cancer cell line .The new antigen proteins may be provided for the further research in the tumor biology,the diagnosis and the cure of the cancer and the preparation of new tumor vaccine.