中华耳科学杂志Issue(1)：136-141,6.DOI:10.3969/j.issn.1672-2922.2014.01.032
MITF基因重组真核细胞表达质粒的构建、表达及意义
Generation and expression of recombinant eukaryotic expression plasmids of MITF gene and its significance
摘要
Abstract
Objective To study exogenous expression and subcellular localization of wild type (WT) and mutant MITF proteins in vitro by generating their expression plasmids for further study of pathogenesis of Waardenburg syndrome (WS). Methods The plasmids pCMV-MITF and pCMV-Flag were ligased after they were cut by double enzyme digestion using mo-lecular cloning technique to generate recombinant eukaryotic expression plasmid pCMV-MITF-Flag, which was as a template to generate expression plasmids pCMV-R217I-Flag and pCMV-T192fs-Flag of novel mutations R217I and T192fs of MITF gene. All constructs were verified by direct nucleotide sequencing. The melanoma UACC903 cells or NIH3T3 cells were trans-fected transiently with the expression plasmids of MITF、R217I and T192fs respectively. The exogenous expression of WT MITF protein and mutant R217I、T192fs proteins were analysed using Western blot assay, while their subcellular distribution were observed using immunofluorescence assay. Results The DNA sequences of expression plasmids of MITF and its mutant R217I、T192fs were correct. Both WT and mutant MITF proteins were detected at the expected size. WT MITF and R217I pro-teins were only localized in the nucleus, whereas T192fs protein showed aberrant localization only in cytoplasm. Conclusion We successfully generated the recombinant eukaryotic expression plasmids of MITF gene and its mutants and drew prelimi-nary conlusion of gene mutation having effect on subcellular distribution of WT MITF proteins in vitro, which lays experimen-tal basis for further study of the moceluar mechanism of WS caused by MITF gene mutations in China.关键词
Waardengurg综合征/MITF/基因突变/分子克隆技术/体外实验Key words
Waardenburg syndrome/MITF/Gene mutation/Molecular cloning technique/In vitro分类
医药卫生引用本文复制引用
张华,冯娟,李家大,罗浑金,陈红胜,梅凌云,贺楚峰,冯永..MITF基因重组真核细胞表达质粒的构建、表达及意义[J].中华耳科学杂志,2014,(1):136-141,6.基金项目
国家自然科学基金（81260160，81170923）资助 （）
