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

目的通过构建基因MITF及其突变体表达质粒初步研究其外源性表达和定位表达,为研究Waardenburg综合征(Waardenburg syndrome,WS)发病机制提供一定的实验依据和基础。方法通过分子克隆技术双酶切pCMV-MITF和pCMV-Flag后连接构建MITF基因重组真核细胞表达质粒pCMV-MITF-Flag,以其为模板分别构建MITF基因新发突变R217I和T192fs表达质粒pCMV-R217I-Flag和pCMV-T192fs-Flag,DNA测序鉴定。MITF、R217I和T192fs表达质粒分别瞬时转染NIH3T3细胞或黑色素瘤UACC903细胞,Western blot和细胞免疫荧光分别检测和观察野生MITF蛋白和突变R217I、T192fs蛋白的表达和分布。结果 MITF及其突变体R217I和T192fs表达质粒经DNA测序鉴定序列正确,三者在黑色素瘤UACC903细胞中正确表达,MITF和R217I仅在细胞核中分布,而T192fs仅在细胞质分布。结论成功构建了MITF基因及其突变体真核细胞表达质粒,突变对MITF蛋白的亚细胞定位产生影响,为在体外实验进一步研究国人MITF基因突变致WS发病的分子机制奠定了实验基础。

Objective To study exogenous expression and subcellular localization of wild type （WT） and mutant M1TF 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-FIag, 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 subeellular 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 R2171 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.