Screening a Novel Human Breast Cancer-Associated Antigen from a ...

[Abstract]

Background & ObjectiveImmunotherapy is a promising new approach in breast cancer treatment, complementing surgery, chemotherapy, radiation and antihormonal therapy. Recent advances have demonstrated the clinical utility of specific monoclonal antibodies that recognize tumor-associated antigens on the surface of the tumor cell in the treatment of breast cancer and B cell lymphoma in humans. Breast cancer-associated tumor antigens take an important role in diagnosis and therapy of mammary carcinoma. Their purposes include early detection of primary tumors, prediction of disease extent and prognosis, early detection of recurrent diseases, evaluation of therapeutic responses, monitoring patient outcome, and differential diagnosis of metastatic tumors of unknown origin. Therefore, finding novel tumor-associated antigens has become the focus of breast cancer-specific immunotherapies.In our previous work, we immunized mice with an extraction of breast cancer tissue, and then made a specific mouse anti-breast cancer monoclonal antibody named M4G3 using hybridism technology, which was shown to have great specificity. This monoclonal antibody has been used for diagnosis and localization of breast cancer clinical trials, which has had ideal effect. The use of M4G3 inspired our interest to explore the nuclear mechanism of the antigen. Thus, the focus of this research is to clone the antigen, and clear its mechanism of action for further investigation of its function and clinical using.MethodsIn the first part, we constructed a ? zap cDNA expression library of breast cancer cell T47D, and screened it by M4G3 as probes to clone the antigen. The positiveclones were subcloned and identified by homologous comparison using BLAST. In addition, we identified the location of positive clone gene in the gene using software. In the second part, several pairs of primers were designed according to the sequence of positive clones using the software of oligo 6.0. Using these primers we detected the expression of positive clone gene in the breast cancer cells and tissues by RT-PCR. Then we studied the positive clones by Western blot, immunocytochemistry and immunofluorescence.ResultsThe result of the first part:1. The A. zap cDNA expression library had 1.2 X 106 independent clones. Its recombination efficiency was 96.9%. The size of cDNA fragments was ranging from 500bp to 2500bp.2. Fourteen positive clones were isolated following three rounds of immunoscreening and the length of cDNA insert was 1700bp~2300bp.3. BLAST homology search revealed that all of the sequences were homologous with Mycoplasma pulmonis and coded two unknown proteins of Mycoplasma pulmonis (AL445563, AL445565) in the GeneBank.The result of the second part:1. The results of RT-PCR show positive signals were detected in T47D cell using three pairs of primers according to the sequence of Mycoplasma pulmonis, but the MDA-MB231, MDA-MB435 and 75 breast cancer tissues were negative.2. MDA-MB231 cell and MDA-MB435 cell, which have no Mycoplasma pulmonis expression, show negative reaction by Western blot with M4G3, but positive by immunofluorescence. While T47D cell, which was identified having Mycoplasma pulmonis expression by RT-PCR, had positive reaction using both two methods.3. The positive rate of immunocytochemistry using M4G3 in 30 breast cancer tissue samples was 85% (26/30). But the Western blots of 35 breast cancer tissues were all negative.Conclusion1. The cDNA library we constructed has an excellent quality and lays solid foundation for screening. In addition, the library is also used for screening other breast cancer relevant genes because it has entire information of this breast cancer cell line.2. The positive clones obtained by screening the cDNA library with M4G3 are homologous with Mycoplasma pulmonis and code two unknown proteins of Mycoplasma pulmonis (AL445563, AL445565) in the GeneBank.3. We have detected Mycoplasma pulmonis from the breast cancer cell T47D. But we have not found Mycoplasma pulmonis from MDA-MB231, MDA-MB435 cell and breast cancer tissues.4. According to the results of Western blot, immunocytochemistry and immunofluorescence, we educe that there are two antigen epitopes that can be recognized by M4G3. One is complicatedly conformational epitope of M4G3-associated antigen, and the other is linear epitope of Mycoplasma pulmonis. The epitope of M4G3-associated antigen is probably complicatedly conformational epitope. When the three-dimensional frame was destroyed using harsh denaturing method, it cannot be recognized by M4G3. So we cannot screen the M4G3-associated antigen using these methods.

Title: Screening a Novel Human Breast Cancer-Associated Antigen from a cDNA Expression Library and Primary Characteristic Study

Category: Tumor Biol

Filename: Screening a Novel Human Breast Cancer-Associated Antigen from a cDNA Expression Library and Primary Characteristic Study.pdf

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