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Cloning and Expression of a Novel MAGE-C2 (Melanoma Associated Antigen) Gene
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Abstract:
Objective: To clone and express human MAGE-C2 gene and to investigate the expression pattern in transfected eukaryote cells. Methods: MAGE-C2 cDNA was amplified by RT-PCR from total RNA of human colorectal adnocarcinoma cell line SW480. Expression vectors of complete open reading frame (ORF) sequence of MAGE-C2 were constructed by PCR and gene cloning technique. After sequencing, the vectors were transfected into E. coli BL21 and 293T cell, respectively. Recombinant GST-MAGE-C2 fusion protein was expressed via IPTG induction. GST-MAGE-C2 fusion protein was purified through glutathione agarose column. Results:The sequence of cloned MAGE-C2 was identical with that reported in GenBank. GFP-MAGE-C2 fusion protein was localized on nuclear identified by fluorescent microscope. SDS-PAGE and Western blot analysis that purified GST-MAGE-C2 fusion protein exhibited a band with Mr. 70 000. Conclusion: The MAGE-C2 gene was cloned and expressed successfully, which not only provides the immunogen for further preparation of anti-MAGE-C2 antibodies, but also applies to research the mechanism of tumor's pathogenesis and cellular immunity response to MAGE.
Keywords:
MAGE-C2 ; RT-PCR ; vector construction ; gene
