听力学及言语疾病杂志
聽力學及言語疾病雜誌
은역학급언어질병잡지
JOURNAL OF AUDIOLOGY AND SPEECH PATHOLOGY
2014年
1期
67-72
,共6页
张华%李家大%罗浑金%陈红胜%梅凌云%贺楚峰%冯永
張華%李傢大%囉渾金%陳紅勝%梅凌雲%賀楚峰%馮永
장화%리가대%라혼금%진홍성%매릉운%하초봉%풍영
Waardengurg综合征%PAX3%基因突变%体外实验
Waardengurg綜閤徵%PAX3%基因突變%體外實驗
Waardengurg종합정%PAX3%기인돌변%체외실험
Waardenburg syndrome%PAX3%Gene mutation%In vitro
目的:通过构建PAX3基因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Waar-denburg综合征(Waardenburg syndrome ,WS)发病机制提供实验基础。方法通过分子克隆技术双酶切pECE -PAX3和pcDNA3.0-HA后连接构建PAX3基因重组真核细胞表达质粒pcDNA3.0-PAX3-HA ,以其为模板分别构建PAX3基因新发突变H80D和H186fs表达质粒pcDNA3.0-H80D-HA和pcDNA3.0-H186fs-HA , DNA测序鉴定。将PAX3、H80D和H186fs表达质粒分别瞬时转染小鼠胚胎成纤维细胞(NIH3T3细胞),采用Western blot和细胞免疫荧光法分别检测和观察野生PAX3蛋白和突变 H80D、H186fs蛋白在NIH3T3细胞中的表达和分布。结果 PAX3及其突变体 H80D和H186fs表达质粒经DNA测序鉴定序列正确,三者在NIH3T3细胞中正确表达,H80D与PAX3仅在细胞核中分布,H186fs在细胞质与细胞核中均有分布。结论本研究成功构建了PAX3基因及其突变体真核细胞表达质粒,突变对PAX3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PAX3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PAX3基因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Waar-denburg綜閤徵(Waardenburg syndrome ,WS)髮病機製提供實驗基礎。方法通過分子剋隆技術雙酶切pECE -PAX3和pcDNA3.0-HA後連接構建PAX3基因重組真覈細胞錶達質粒pcDNA3.0-PAX3-HA ,以其為模闆分彆構建PAX3基因新髮突變H80D和H186fs錶達質粒pcDNA3.0-H80D-HA和pcDNA3.0-H186fs-HA , DNA測序鑒定。將PAX3、H80D和H186fs錶達質粒分彆瞬時轉染小鼠胚胎成纖維細胞(NIH3T3細胞),採用Western blot和細胞免疫熒光法分彆檢測和觀察野生PAX3蛋白和突變 H80D、H186fs蛋白在NIH3T3細胞中的錶達和分佈。結果 PAX3及其突變體 H80D和H186fs錶達質粒經DNA測序鑒定序列正確,三者在NIH3T3細胞中正確錶達,H80D與PAX3僅在細胞覈中分佈,H186fs在細胞質與細胞覈中均有分佈。結論本研究成功構建瞭PAX3基因及其突變體真覈細胞錶達質粒,突變對PAX3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PAX3基因突變緻WS髮病的分子機製奠定瞭實驗基礎。
목적:통과구건PAX3기인급기돌변체표체질립초보연구기외원성표체화정위표체,위연구Waar-denburg종합정(Waardenburg syndrome ,WS)발병궤제제공실험기출。방법통과분자극륭기술쌍매절pECE -PAX3화pcDNA3.0-HA후련접구건PAX3기인중조진핵세포표체질립pcDNA3.0-PAX3-HA ,이기위모판분별구건PAX3기인신발돌변H80D화H186fs표체질립pcDNA3.0-H80D-HA화pcDNA3.0-H186fs-HA , DNA측서감정。장PAX3、H80D화H186fs표체질립분별순시전염소서배태성섬유세포(NIH3T3세포),채용Western blot화세포면역형광법분별검측화관찰야생PAX3단백화돌변 H80D、H186fs단백재NIH3T3세포중적표체화분포。결과 PAX3급기돌변체 H80D화H186fs표체질립경DNA측서감정서렬정학,삼자재NIH3T3세포중정학표체,H80D여PAX3부재세포핵중분포,H186fs재세포질여세포핵중균유분포。결론본연구성공구건료PAX3기인급기돌변체진핵세포표체질립,돌변대PAX3단백적아세포정위산생영향,위재체외실험진일보연구국인PAX3기인돌변치WS발병적분자궤제전정료실험기출。
Objective To study exogenous expression and subcellular localization of wild type (WT ) and mu-tant PAX3 proteins in vitro by generating their expression plasmids for further study of pathogenesis of Waarden-burg syndrome (WS) .Methods The plasmids pECE-PAX3 and pcDNA3 .0-HA were ligased after they were cut by double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pcDNA3 .0-PAX3-HA ,which was as a template to generate expression plasmids pcDNA 3 .0 -H80D -HA and pcDNA3 .0-H186fs-HA of novel mutations H80D and H186fs of PAX3 gene .All constructs were verified by di-rect nucleotide sequencing .NIH3T3 cells were transfected transiently with the expression plasmids of PAX3 ,H80D and H186fs respectively .The exogenous expression of WT PAX3 protein and mutant H80D ,H186fs proteins were analysed using Western blot assay ,while their subcellular distribution were observed using immunofluorescence as-say .Results The DNA sequences of expression plasmids of PAX3 and its mutant H80D ,H186fs were correct . Both WT and mutant PAX3 proteins were detected at the expected size .WT PAX3 and H80D proteins were only lo-calized in the nucleus ,whereas H186fs protein showed aberrant localization in both cytoplasm and nucleus .Conclu-sion We successfully generated the recombinant eukaryotic expression plasmids of PAX 3 gene and its mutants and drew preliminary conlusion of gene mutation having effect on subcellular distribution of WT PAX 3 proteins in vitro , which lays experimental basis for further study of the moceluar mechanism of WS caused by PAX3 gene mutations in China .