CAJ | 학술논문

目的构建pEGFP-N1-CKB真核表达载体并将其稳定转染到肺鳞癌细胞NCI-H520中,建立稳定转染的NCI-H520细胞系,为后续研究奠定基础.方法以人CKB cDNA文库为模板,用PCR扩增人CKB cDNA的编码区,并将扩增的cDNA片段与载体连接后亚克隆到真核表达载体pEGFP-N1中.重组子经酶切分析及测序鉴定后,用脂质体转染技术将其导入到人肺鳞癌细胞系NCI-H520,经G418筛选并建立稳定的转染细胞株,应用Western blot检测转染前后该细胞株CKB基因的表达.结果 pEGFP-N1-CKB经酶切鉴定及DNA测序证实序列完全正确,真核表达载体构建成功；经Western blot检测,重组质粒转染株中CKB基因的表达水平明显高于对照组,证实CKB基因已稳定转染到NCI-H520细胞中并得到表达.结论成功构建了pEGFP-N1-CKB真核表达质粒,建立了稳定表达CKB的NCI-H520细胞系,为进一步研究CKB的生物学功能奠定了基础.
목적 구건pEGFP-N1-CKB진핵표체재체병장기은정전염도폐린암세포NCI-H520중,건립은정전염적NCI-H520세포계,위후속연구전정기출.방법 이인CKB cDNA문고위모판,용PCR확증인CKB cDNA적편마구,병장확증적cDNA편단여재체련접후아극륭도진핵표체재체pEGFP-N1중.중조자경매절분석급측서감정후,용지질체전염기술장기도입도인폐린암세포계NCI-H520,경G418사선병건립은정적전염세포주,응용Western blot검측전염전후해세포주CKB기인적표체.결과 pEGFP-N1-CKB경매절감정급DNA측서증실서렬완전정학,진핵표체재체구건성공；경Western blot검측,중조질립전염주중CKB기인적표체수평명현고우대조조,증실CKB기인이은정전염도NCI-H520세포중병득도표체.결론 성공구건료pEGFP-N1-CKB진핵표체질립,건립료은정표체CKB적NCI-H520세포계,위진일보연구CKB적생물학공능전정료기출.
Objective To construct an eukaryotic expression vector pEGFP-N1-CKB and establish a stably transfected NCI-H520 cell line.Methods Human CKB gene was amplified by PCR with human CKB cDNA library as the template and the fragment was combined with plasmid pEGFP-N1.The recombinant expression vector,pEGFP-N1-CKB,was transfected to NCI-H520 using Lipofectamin.The stably transfected cell line was established after G418 selection and the expression level of CKB gene before and after transfection was detected by Western blot.Results After identification by restriction enzyme digestion and sequencing,the eukaryotic expression vector,pEGFP-N1-CKB,was successfully constructed.The expression level of CKB in NCI-H520 transfected by pEGFP-N1-CKB was significantly higher than that in control.CKB gene had a stable transfection in NCI-H520 cells.Conclusions An eukaryotic plasmid encoding CKB (pEGFP-N1-CKB) has been constructed and a cell line expressed CKB stably has been successfully prepared.