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In this study, pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of N-glycolylneuraminic acid was cloned from pig small intestine and characterized. The open reading frame of pcmah was 1,734 bp encoded 577 amino acids and consists of fourteen exons. Organ expression pattern analysis reveals that pig CMAH mRNA is mainly expressed in pig rectum, tongue, spleen, and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when PK15 cells and ECV304 cells were transfected with the cloned pcmah, NeuGc contents of these transfectants were greater in comparison to vector transfectants used as control. In addition, in the functional analysis of NeuGc, human serum-mediated cytotoxicity was elevated in the ectopic NeuGc expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG were slightly increased indicating that the human IgM type was a major anti-NeuGc antibody. From the results, it was concluded that the pig CMAH gene was capable to synthesize the NeuGc acting as a xenoantigen in human, confirming the NeuGc-mediated rejection response in pig-to-human xenotransplantati