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目的检测人恶性黑色素瘤A375细胞膜上MC1R蛋白及其cDNA,为研究黑色素瘤细胞表面受体及靶向配体奠定基础。方法利用放射配体分析法检测人恶性黑色素瘤A375细胞膜上的MC1R蛋白;用RTPCR方法扩增人恶性黑色素瘤A375细胞MC1R的cDNA,并克隆至pMD18T质粒,进行酶切分析鉴定及扩增片段DNA序列测定。结果放射配体分析结果表明,125IαMSH可与人恶性黑色素瘤A375细胞膜特异结合。RTPCR结果显示,扩增产物的大小与MC1R的cDNA相符。测序结果证实扩增片段为MC1R基因的特异片段。结论证实人恶性黑色素瘤A375细胞膜上表达MC1R蛋白。MC1R基因的成功克隆为其进一步研究提供了必要条件。
Objective To detect the expression of MC1R protein and its cDNA on the human melanoma A375 cell membrane, and lay a foundation for the study of melanoma cell surface receptors and targeting ligands. Methods The expression of MC1R protein in human melanoma A375 cell line was detected by radioligand analysis. The cDNA of MC1R in human melanoma A375 cells was amplified by RTPCR and cloned into pMD18T plasmid for enzyme digestion analysis to identify and amplify the fragment DNA Sequencing. Results Radioligand analysis showed that 125IαMSH could specifically bind to human melanoma A375 cell membrane. The results of RTPCR showed that the size of amplified product was consistent with that of MC1R. Sequencing results confirmed that the amplified fragment was a specific fragment of MC1R gene. Conclusion The MC1R protein was expressed on human melanoma A375 cell membrane. The successful cloning of MC1R gene provided the necessary conditions for its further study.