Gene Cloning, Expression And Distribution Of Melanoma Antigen MAGE-A10

Melanoma antigen genes (MAGEs) were a number of gene families cloned from Melanoma, which expressed specifically on some types of cancers but not on normal tissue except for testis and placenta. Most members of MAGE families can induce specific CTL response in host, so they have potential value in the immunotherapy of tumors.MAGE-A10 belongs to MAGE-A subfamily. The full length of cDNA was 2.5kb contained 4 exons, coding a protein with Mr of 72kDa. MAGE-A 10 was highly expressed on tumor cells with a high immunogenesity. A HLA-A2 restricted nona-peptide 254GLYDGMEHL262 coded by MAGE-A10 could elicit specific CTL response which was found by tetramer technology recently. Based on highly frequency of HLA-A2 in Asians, it is important to investigate the expression, function and potential immunotherapy value of this molecule. To explore the expression of MAGE-A 10 and distribution of that in cancers, the following works have been done in our department:1. Cloning and prokaryotic expression of human MAGE-A10 cDNAAims: To clone human MAGE-A10 cDNA, construct the recombination expression vector, and express its recombinant protein in E. coli. Methods: The cDNA encoding human MAGE-A 10 gene was amplified by RT-PCR from human melanoma cell line LiBr. Then the MAGE-A 10 gene was inserted into plasmids pMD18-T. After sequencing, the MAGE-A 10 was cloned into the prokaryotic expression vector pGEX-4T-3 to construct the recombinantexpression vector pGEX-4T-3-MAGE-A10 and were transformed into E. coli BL21.The recombinant GST-MAGE-A10 fusion protein was expressed under induction of IPTG. GST-MAGE-A10 fusion protein was purified through glutathione agarose column. Results: The sequence of MAGE-AIO was identical to that from GenBank. Affinity column purified-GST-MAGE-A10 fusion protein appeared a band of Mr. 70KD on SDS-PAGE, and the antigenic specificity of the fusion protein was confirmed by Western blot. Conclusions: The MAGE-A10 gene was cloned and expressed successfully, which not only provided the immunogen for preparation of anti-MAGE-A10 antibody but also were useful in research work on the mechanism of tumor pathogenesis and cellular immunological response to MAGE molecules.2. Preparation and identification of monoclonal antibodies against human MAGE-AIOAims: To prepare and identify the monoclonal antibody to MAGE-A10 Methods: GST-MAGE-A10 fusion protein was largely prepared and purified. Then BALB/c mice were immunized with purified-GST-MAGE-A10 fusion protein to get anti-MAGE-A10 antibody. Normal testis tissue was used to identify specific mAbs. The anti-MAGE-A10 antibody was purified as follows:Firstly, mAbs from ascites fluid were sedimentated with ammonium sulfate,then desalted by Sepharose G-25 column and purified by anion exchange chromatography on Sepharose Q Fast Flow column using FPLC (Phamarcia Amercia). Results: 19 hybridoma cell lines secreting mAbs against MAGE-A10 have been established. One of them named LX-MAGE-A10.6 was employed in immunohistochemical staining with highly specific and sensitivity. Conclusions: We got one hybridoma cell which could be useful for investigation of its expression and distribution.3. The expression patterns of MAGE-A10 in tumorsAims: To explore the expression and the clinical significance of MAGE-A10 in human lung cancer and other four types of digestive system cancers. Methods: By using tissue array and S-P immunohistochemical methods, the expression of MAGE-A10 protein in lung cancer and four types of digestive system cancers was investigated and the correlation of MAGE-A10 expression with histological types and pathological grades was also evaluated. Results: (1)The positive expression rates of MAGE-A10 were 36.4% in small cell lung carcinoma(SCLC), 76.5% in squamous cell lung carcinoma and 40.0%in lung adenocarcinoma, respectively, whereas no positive case can be found in large cell lung carcinoma and bronchioloalveolar carcinoma. There was no statistical significance between the expression of MAGE-A10 protein in SCLC and that in NSCLC. The...