CAJ | 학술논문

目的克隆人细胞周期蛋白D1基因,构建真核表达载体,诱导并鉴定其表达.方法利用RT-PCR法,以低分化鼻咽癌CNE-22细胞mRNA为模板,扩增周期蛋白D1基因,构建重组装达载体pIRES2-EGFP-D1和pEGFP-C2-D1,用脂质体Lipofectamine~(TM) 2000将重组质粒转染到低分化鼻咽癌CNE-22细胞,诱导其表达,免疫荧光、Western blotting等鉴定其表达.结果电泳获得918 bp预期片段,重组质粒经酶切、PCR和测序分析证实含有周期蛋白D1完整读码框,将重组质粒导人CNE-2Z细胞并成功表达,目的蛋白经免疫荧光和Western blotting鉴定具有生物学活性.结论成功克隆并构建含周期蛋白D1基因的真核表达GFP质粒,为进一步研究周期蛋白D1在鼻咽癌CNE-22细胞中的作用奠定基础.
목적 극륭인세포주기단백D1기인,구건진핵표체재체,유도병감정기표체.방법 이용RT-PCR법,이저분화비인암CNE-22세포mRNA위모판,확증주기단백D1기인,구건중조장체재체pIRES2-EGFP-D1화pEGFP-C2-D1,용지질체Lipofectamine~(TM) 2000장중조질립전염도저분화비인암CNE-22세포,유도기표체,면역형광、Western blotting등감정기표체.결과 전영획득918 bp예기편단,중조질립경매절、PCR화측서분석증실함유주기단백D1완정독마광,장중조질립도인CNE-2Z세포병성공표체,목적 단백경면역형광화Western blotting감정구유생물학활성.결론성공극륭병구건함주기단백D1기인적진핵표체GFP질립,위진일보연구주기단백D1재비인암CNE-22세포중적작용전정기출.
Objective To construct the eukaryotic expression vectors of human cyclin D1 gene and express them in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells). Methods The full-length cyclin D1 was cloned from CNE-2Z cells by RT-PCR. The cDNA fragments were inserted into pIRES2-EGFP plasmids and pEGFP-C2 plasmids and confirmed by restriction enzyme digestion, PCR and sequencing. The recombinant vectors were ttansfected into CNE-2Z cells via Lipofectamine~(TM) 2000, and the expression of cyclin D1 in the cells was examined by immunofluorescence and Western blotting. Results Agarose gel electrophoresis showed a 918 bp band of the RT-PCR products, which matched the expected size. Restriction enzyme digestion, PCR and sequencing demonstrated successful construction of the recombinant vectors. CNE-2Z cells transfected with the recombinant vectors expressed cyclin D1 protein or cyclin D1-GFP protein as were verified by immunofluorescence and Western blotting. Conclusion We have cloned cyclin D1 gene and constructed its eukaryotic expression vectors that can be expressed in nasopharyngeal carcinoma cells, which may facilitate the study of the role of cyclin D1 in the development of nasopharyngeal carcinoma.