| ObjectiveFrom 1990s, the successful cloning of many tumor antigen genes of human kind has driven the development of tumor immunology enormously. And so does it to the application of tumor immunodiagno sties and immunotherapy. It has become a hot point to search for a target molecule with strong specificity and definite gene function and biochemical mechanism. In recent years, the incidence of breast cancer has been increasing gradually. In multiple studies of specific antigen markers of breast cancer, there isn't any new antigen with high specificity and sensitivity except the rather specific c-erbB-2. In the paper, with M4G3 monoclonal antibody to breast cancer prepared in our hospital previously, we have successfully accomplished the cloning and expressing of one specific antigen gene in breast cancer by construction of cDNA library and screening techniques. We believe that it will provide reliable proof for clinical usage of this antibody and dependable evidence for profound establishment of the mechanisms in formation and progression of breast cancer.Methods:In the first part, we survived and cultured M4G3 hybridoma in vitro. After two weeks of injecting hybridoma into mouse abdominal cavity, we collected abundant ascites. At the same time, 4 strains of breast cancer cells had been growing in vitro , digested and collected for immunocytochemistry and Western blotting with M4G3 ascites.In the second part, we constructed a cDNA expression library of breast cancer cell T47D with gt11 as vector. Then the recombinant DNA was packaged in vitro. . Identification of cDNA library was followed, such as clone efficiency, recombinant efficiency, library integrality and the size of inserts. Finally the library was amplified .In the third part, we used M4G3 ascites as probe to screen the library for obtaining positive clone. Once it was found, we would go on sequencing and analyzing at web NCBI / BLAST.ResultThe result of the first part:1. Immunocytochemistry reveals that breast cancer cells of T47D and MDA-MB-231 have positive reaction. Positive particles position cell membrance and cell plasma respectively. The other two cells MCF-7 and MDA-MB-43 5 are negative.2. Western blotting indicate that T47D and MDA-MB-231 are also positive and the molecule weight of positive protein is estimated as 48,000 dalton. MCF-7 and MD A-MB-43 5 are also negative.The result of the second part:1. The unamplified cDNA library consists of 1.0 106 independent clones in which the percentage of recombinant clones is about 96.7%. The average size of inserts is 1.0kb.2. PCR test that -actin is included in the library and the titer of amplified cDNA library is 1.0 1010.The result of the third part:1. One positive clone has been gained from the library screened by M4G3 McAb. The cDNA of the clone is 3.3 kb length.2. The positive clone has been purified and sent to Takara Company for sequencing.Conclusion1. The breast cancer antigen specifically combined with M4G3 McAb has 48,000 dalton molecule weight. This antigen is different from ER, PR, BP1 and P185.2. These results show that cDNA expression library has an excellent quality and lays solid foundation for further screening. In addition, the library is also used for screening other breast cancer relevant genes.3. The length of cDNA obtained from positive clone is about 3.3 kb and sequence analysis is under way. |
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