中华耳科学杂志
中華耳科學雜誌
중화이과학잡지
CHINESE JOURNAL OF OTOLOGY
2014年
1期
136-141
,共6页
张华%冯娟%李家大%罗浑金%陈红胜%梅凌云%贺楚峰%冯永
張華%馮娟%李傢大%囉渾金%陳紅勝%梅凌雲%賀楚峰%馮永
장화%풍연%리가대%라혼금%진홍성%매릉운%하초봉%풍영
Waardengurg综合征%MITF%基因突变%分子克隆技术%体外实验
Waardengurg綜閤徵%MITF%基因突變%分子剋隆技術%體外實驗
Waardengurg종합정%MITF%기인돌변%분자극륭기술%체외실험
Waardenburg syndrome%MITF%Gene mutation%Molecular cloning technique%In vitro
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。
目的:通過構建基因MITF及其突變體錶達質粒初步研究其外源性錶達和定位錶達,為研究Waardenburg綜閤徵(Waardenburg syndrome,WS)髮病機製提供一定的實驗依據和基礎。方法通過分子剋隆技術雙酶切pCMV-MITF和pCMV-Flag後連接構建MITF基因重組真覈細胞錶達質粒pCMV-MITF-Flag,以其為模闆分彆構建MITF基因新髮突變R217I和T192fs錶達質粒pCMV-R217I-Flag和pCMV-T192fs-Flag,DNA測序鑒定。MITF、R217I和T192fs錶達質粒分彆瞬時轉染NIH3T3細胞或黑色素瘤UACC903細胞,Western blot和細胞免疫熒光分彆檢測和觀察野生MITF蛋白和突變R217I、T192fs蛋白的錶達和分佈。結果MITF及其突變體R217I和T192fs錶達質粒經DNA測序鑒定序列正確,三者在黑色素瘤UACC903細胞中正確錶達,MITF和R217I僅在細胞覈中分佈,而T192fs僅在細胞質分佈。結論成功構建瞭MITF基因及其突變體真覈細胞錶達質粒,突變對MITF蛋白的亞細胞定位產生影響,為在體外實驗進一步研究國人MITF基因突變緻WS髮病的分子機製奠定瞭實驗基礎。
목적:통과구건기인MITF급기돌변체표체질립초보연구기외원성표체화정위표체,위연구Waardenburg종합정(Waardenburg syndrome,WS)발병궤제제공일정적실험의거화기출。방법통과분자극륭기술쌍매절pCMV-MITF화pCMV-Flag후련접구건MITF기인중조진핵세포표체질립pCMV-MITF-Flag,이기위모판분별구건MITF기인신발돌변R217I화T192fs표체질립pCMV-R217I-Flag화pCMV-T192fs-Flag,DNA측서감정。MITF、R217I화T192fs표체질립분별순시전염NIH3T3세포혹흑색소류UACC903세포,Western blot화세포면역형광분별검측화관찰야생MITF단백화돌변R217I、T192fs단백적표체화분포。결과MITF급기돌변체R217I화T192fs표체질립경DNA측서감정서렬정학,삼자재흑색소류UACC903세포중정학표체,MITF화R217I부재세포핵중분포,이T192fs부재세포질분포。결론성공구건료MITF기인급기돌변체진핵세포표체질립,돌변대MITF단백적아세포정위산생영향,위재체외실험진일보연구국인MITF기인돌변치WS발병적분자궤제전정료실험기출。
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.