Cloning, Expression And Identification Of Human Melanoma Antigen MAGE-A9 In Hepatocellular Carcinomas

The occurrence of HCC is high in China. However, there has no any satisfied treatment against hepatocellular carcinoma. A potentially useful strategy for treating HCC is to develop vaccines against antigens that are commonly expressed on this type of cancer. It has been reported that the specific expression of some MAGE-A genes in HCC is high. Therefore, the tumor-specific expression has made the MAGE-A genes interesting as candidates for anti hepatocellular carcinoma immunotherapy.In 1991, van der Bruggen et al. identified the first melanoma antigen gene, MAGE-A1, in a human melanoma cell line. Since then, 12 MAGE-A genes have been isolated and sequence homology. Genes of MAGE-A family are expressed in several types of solid tumors but are silent in normal tissues with the exception of male germline cells. Therefore, peptides encoded by MAGE-A genes are strictly tumor-specific antigens that can be recognized by CTL and constitute promising targets for diagnosis and therapy. MAGE-A9 is of particular relevance in the context of the MAGE-A gene family, which has been mapped to chromosome X, and its gene product has been identified as a protein of an apparent molecular weight of Mr 35,000. Since little is known about the expression of MAGE-A9 in hepatocellular carcinomas in Chinese population, we examined the expression of MAGE-A9 in 39 prognostic parameters of hepatocellular carcinoma. In this study, we fulfilled some objects. Objectives:1. To extract total RNA of MAGE-A9 gene from hepatocelllar carcinoma tissue, to amplify the MAGE-A9 cDNA products and to subclone it into the T-vector for DNA sequencing analysis and identifying the sequence.2. To construct the prokaryotic expression system of MAGE-A9 gene, to identify and purify the culture products.3. To generate the monoclonal antibody of MAGE-A9 antigen4. To examine the expression and location of MAGE-A9 in HCCMethods1. Total RNA was extracted from the hepatocellular carcinoma tissue by using Trizaol and reverse transcribed. MAGE-A9 cDNA was amplified by PCR, ligated with a similarly restricted pMD-18T simple vector, sequceced and identified after comparing with GenBank.2. The purified plasmid was digested with EcoR â…  and Hind â…¢ and subcloned into expression plasmid vector pBAD/gâ…¢ with six-histidine tag and the recombinant plasmid was transferred into E.coli TOP 10. This Culture was induced by L-arabinose and pufified by HiTrap affinity columns. SDS-PAGE and Western blot monitored the production and purification of the recombinant protein.3. BALB/c mice were immunized with purified material. Animals were sacrificed, and fusions were performed as described in manuscript. Hybridoma supernatants were screened by ELISA and the purified MAGE-A9 protein was used as the antigen. The monoclonal antibody was identified by SDS-PAGE and Dot blot.4. The wxpression and location of MAGE-A9 in HCC were examined through Immunohistochemical assay.Results1. The 945bp MAGE-A9 gene was amplified and its sequence product was same to MAGE-A9 cDNA published on GenBank2. Restriction mapping of recombinant plasmid revealed that the cloning vector pMD18-T - MAGE-A9 was successfully constructed. L-arabinose induction of the expression system pBAD/gIII-MAGE-A9 resulted in the production of a protein exhibiting an apparent Mr 35,000, matching the previously described MAGE-A9 gene product and identified by Western blot. Purified products were generated by using HiTrap column.3. The positive rate of ELISA was 100% after three subclones and a stable hybridoma cell line that cans secret anti-MAGE-A9 monoclonal antibody has been created and then been named AM9. The results of Dot blot revealed that the cell line secreting anti-MAGE-A9 monoclonal antibody showed exclusive reactivity on the MAGE-A9 protein.4. Intracellular detection of MAGE-A9 gene product was attempted in hepatocellular carcinoma tissues and a cytoplasmic reaction was obtained with anti-MAGE-A9 monoclonal antibody. Tissues of 39 hepatocellular carc...