重组人源MPG基因真核表达载体构建及转染非小细胞肺癌多药耐药细胞的研究

Construction of human MPG eukaryotic expression vector and expression in human non small cell lung cancer multidrug resistance cell line

目的构建人N甲基化嘌呤DNA糖基化酶(NmethylpurineDNAglycosylase,MPG)基因真核表达载体,并研究其在稳定转染的人非小细胞肺癌多药耐药细胞中的表达情况。方法应用分子克隆技术构建pCMVScript/MPG重组真核表达载体;应用脂质体介导的基因转染技术将其导入人非小细胞肺癌多药耐药细胞株A549/CDDP内;应用G418筛选稳定表达的转染细胞;应用PCR检测pCMVScript载体上的NEOr基因,应用RTPCR及Westernblot检测MPG基因的表达。结果构建产物经限制性酶切分析及基因序列测定证实为pCMVScript/MPG重组表达载体;转染pCMVScript/MPG载体组(MP组)及转染pCMVScript载体组(P组)细胞内检测到NEOr基因,未转染组(C组)则未检测到;MP组细胞内MPGmRNA水平较P组及C组细胞显著增高(P<0.01);MP组细胞内MPG蛋白质水平较P组及C组细胞显著增高(P<0.01)。结论成功地构建了人MPG基因表达载体,并在人非小细胞肺癌多药耐药细胞A549/CDDP获得了稳定、高效的表达。

Objective To construct human N-methylpurine glycosylase （MPG） eukaryotic expression vector and explore its expression levels in stable transfected human non small cell lung cancer multidrug re- sistance cell line. Methods Recombinant eukaryotic expression vector pCMV-Script/MPG was constructed by moleculer cloning technique, then transfected into human non small cell lung cancer muhidrug resistance cell line A549/CDDP by Lipofectamine 2000. In the stable transfected cells which were screened out by G418, NEO^τ gene on pCMV-Script vector was detected by PCR and the expression levels of MPG were determined by RT-PCR and Western blotting. Results The MPG eukaryotic expression vector was manifested by restriction endonuclease digestion and gene sequencing. The PCR product of NEO^τ gene was detected in cells transfected with pCMV-Script/MPG vector or pCMV-Script vector. The mRNA and level of MPG was higher in cells trans fected with pCMV-Script/MPG vector （5.11 ± 0.1 ） than that in cells transfected with pCMV-Script vector （2. 02 ±0.08 ） and without transfection （2.00± 0.2） （ P 〈 0.01 ） , so did the protein level of MPG. Conclusion Human MPG eukaryotic expression vector was constructed successfully and expressed in a stable and high efficiency manner in human non small cell lung cancer multidrug resistance cell line A549/CDDP.