重组GSTP1真核表达质粒的构建及稳定转染NCI-H520细胞系的建立

Construction of eukaryotic expression plasmid of human GSTP1 and establishment of NCI-H520 cell line stably transfected with the constructed plasmid

目的构建pcD N A3.1(+)/G STP1真核表达载体并将其稳定转染到肺鳞癌细胞N C I-H 520中,建立稳定转染的N C I-H 520细胞系,为后续研究奠定基础。方法以pD N R-LIB-G STP1为模板,用PC R扩增G STP1 cD N A的编码区,并将扩增的cD N A片段与载体连接后亚克隆到真核表达载体pcD N A3.1(+)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N C I-H 520,经G 418筛选并建立稳定的转染细胞株,应用W estern blotting检测转染前后该细胞株G STP1基因在蛋白水平的表达。结果 pcD N A3.1(+)/G STP1经酶切鉴定及D N A测序证实序列完全正确,真核表达载体构建成功;经W estern blotting检测,重组质粒转染株中G STP1基因的蛋白表达水平明显高于对照组,证实G STP1基因已稳定转染到N C I-H 520细胞中并得到表达。结论成功构建了pcD N A3.1(+)/G STP1真核表达质粒,建立了稳定表达G STP1的N C I-H 520细胞系,为进一步研究G STP1的生物学功能奠定了基础。

【Objective】To construct an eukaryotic expression vector pcDNA3.1（＋） /GSTP1 and establish a stably transfected NCI-H520 cell line,lay the groundwork for future study.【Methods】Human GSTP1 gene was amplified by PCR with pDNR-LIB-GSTP1 as the template and the fragment was combined with plasmid pcDNA3.1（＋）.The recombinant expression vector,pcDNA3.1（＋） /GSTP1,was transfected to NCI-H520 using Lipofectamin.The stably transfected cell line was established after G418 selection and the expression level of GSTP1 gene before and after transfection was detected by Western blotting.【Results】After identification by restriction enzyme digestion and sequencing,the eukaryotic expression vector,pcDNA3.1（＋） /GSTP1,was successfully constructed.The expression level of GSTP1 in NCI-H520 transfected by pcDNA3.1（＋） /GSTP1 was significantly higher than that in the control.GSTP1 gene had a stable transfection in NCI-H520 cells.【Conclusion】An eukaryotic plasmid encoding GSTP1（pcDNA3.1（＋） /GSTP1） has been constructed and a cell line expressing GSTP1 stably has been prepared successfully.