Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE...Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results：The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 （21%） hepatocellular carcinoma specimens. Conclusion：MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.展开更多
Objective: Melanoma antigen genes(MAGE) genes have been found in many kinds of tumor tissue, but not in normal tissue except testis and placentas. The Ags encoded by MAGE genes therefore are strictly tumor-specific. T...Objective: Melanoma antigen genes(MAGE) genes have been found in many kinds of tumor tissue, but not in normal tissue except testis and placentas. The Ags encoded by MAGE genes therefore are strictly tumor-specific. The most current researches associated with these genes focus on the tumor vaccination using these Ags. Few reports are concerning these genes' functions. In this study, we investigated the role of MAGE-A1 gene on NIH3T3 cells after transferring with it. Methods: Clone the MAGE-A1 into the plasmids pEGFP-C3 and pcDNA3.1, then transfer the reconstructed plasmids and primary plasmids into the NIH3T3 cells using a new transfer reagent FuGENE 6. Selecting the positively transferred cells by G418. Identified by RT-PCR, Western blot, Immunocytochemistry, Laser Scanning Confocal Microscope and Fluoroscope. The cells mobile ability was measured with Millicell-PCF. The cell cycle and apoptosis were measured with Flow Cytometry. Results:The apoptosis rate of NIH3T3 cells that transferred with control plasmid pcDNA3.1 was 13.4% and the ratios that stay in S phase and G2-M phase were 5.68% and 1.04% respectively. The apoptosis rate of NIH3T3 cells that transferred with pcDNA3.1-A1 was 0.90% and the ratios that stayed in S phase and G2-M phase were 19.31% and 13.47% respectively. The apoptosis rate of the cells that transferred with control plasmid pEGFP-C3 was 1.87%, a little higher than 1.47% of those transferred with pEGFP-C3-A1. Conclusion:The MAGE-A1 gene may enhance the cell cycle, inhibit the apoptosis and raise the mobile (ability) of NIH3T3 cells.展开更多
Objective To investigate the expression of the preferentially expressed antigen of melanoma（PRAME） gene in acute leukemia and its clinical significance. Methods The level of expressed PRAME mRNA in bone marrow monon...Objective To investigate the expression of the preferentially expressed antigen of melanoma（PRAME） gene in acute leukemia and its clinical significance. Methods The level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia（AL） and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR.Correlation analyses between PRAME gene expression and the clinical characteristics（gender,age,white blood count,immunophenotype of leukemia,percentage of blast cells, and karyotype） of the patients were performed. Results The PRAME gene was expressed in 38.2%of all 34 patients,in 40.7%of the patients with acute myelogenous leukemia （AML,n=27）,and in 28.6%of the patients with acute lymphoblastic leukemia（ALL,n=7）,but was not expressed in the healthy volunteers.The difference in the expression levels between AML and ALL patients was statistically significant.The rate of gene expression was 80%in M3,33.3%in M2,and 28.6%in Ms.Gene expression was also found to be correlated with CD15 and CD33 expression and abnormal karyotype,but not with age,gender,white blood count or percentage of blast cells. Conclusions The PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease.This gene is also a candidate target for the immunotherapy of acute leukemia.展开更多
文摘Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results：The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 （21%） hepatocellular carcinoma specimens. Conclusion：MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.
文摘Objective: Melanoma antigen genes(MAGE) genes have been found in many kinds of tumor tissue, but not in normal tissue except testis and placentas. The Ags encoded by MAGE genes therefore are strictly tumor-specific. The most current researches associated with these genes focus on the tumor vaccination using these Ags. Few reports are concerning these genes' functions. In this study, we investigated the role of MAGE-A1 gene on NIH3T3 cells after transferring with it. Methods: Clone the MAGE-A1 into the plasmids pEGFP-C3 and pcDNA3.1, then transfer the reconstructed plasmids and primary plasmids into the NIH3T3 cells using a new transfer reagent FuGENE 6. Selecting the positively transferred cells by G418. Identified by RT-PCR, Western blot, Immunocytochemistry, Laser Scanning Confocal Microscope and Fluoroscope. The cells mobile ability was measured with Millicell-PCF. The cell cycle and apoptosis were measured with Flow Cytometry. Results:The apoptosis rate of NIH3T3 cells that transferred with control plasmid pcDNA3.1 was 13.4% and the ratios that stay in S phase and G2-M phase were 5.68% and 1.04% respectively. The apoptosis rate of NIH3T3 cells that transferred with pcDNA3.1-A1 was 0.90% and the ratios that stayed in S phase and G2-M phase were 19.31% and 13.47% respectively. The apoptosis rate of the cells that transferred with control plasmid pEGFP-C3 was 1.87%, a little higher than 1.47% of those transferred with pEGFP-C3-A1. Conclusion:The MAGE-A1 gene may enhance the cell cycle, inhibit the apoptosis and raise the mobile (ability) of NIH3T3 cells.
文摘Objective To investigate the expression of the preferentially expressed antigen of melanoma（PRAME） gene in acute leukemia and its clinical significance. Methods The level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia（AL） and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR.Correlation analyses between PRAME gene expression and the clinical characteristics（gender,age,white blood count,immunophenotype of leukemia,percentage of blast cells, and karyotype） of the patients were performed. Results The PRAME gene was expressed in 38.2%of all 34 patients,in 40.7%of the patients with acute myelogenous leukemia （AML,n=27）,and in 28.6%of the patients with acute lymphoblastic leukemia（ALL,n=7）,but was not expressed in the healthy volunteers.The difference in the expression levels between AML and ALL patients was statistically significant.The rate of gene expression was 80%in M3,33.3%in M2,and 28.6%in Ms.Gene expression was also found to be correlated with CD15 and CD33 expression and abnormal karyotype,but not with age,gender,white blood count or percentage of blast cells. Conclusions The PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease.This gene is also a candidate target for the immunotherapy of acute leukemia.
