目的 构建PTEN基因真核表达载体,为PTEN基因功能研究提供工具.方法 从人淋巴细胞中提取总RNA,采用反转录-聚合酶链反应扩增PTEN基因编码区,将其克隆入pEGFP-N1载体中.通过聚合酶链反应、酶切和DNA测序鉴定所构建的载体.脂质体包裹重组载体转染骨肉瘤MG63细胞,RT-PCR和Western blot检测转染后的骨肉瘤MG63细胞中PTEN基因mRNA和蛋白质表达情况.结果 经过限制性酶切和测序鉴定得到重组子GFP-PTEN大小符合,序列与GenBank中人DNA的PTEN基因(NM_000314)完全一致.转染GFP-PTEN基因的骨肉瘤MG63细胞中PTEN基因mRNA和蛋白高水平表达.结论 成功地构建了PTEN基因真核表达载体.%Objective To construct an eukaryotic expression vector for phosphatase and tensin homology deletedon chromosome ten( PTEN ) gene lymphocytes and provide a tool for studying of PTEN gene function. Methods The total RNAs were isolated from human lymphocytes. The cDNA of PTEN gene was amplified by reverse transcription polymerase chain reaction( RT-PCR ). After purification, the gene was cloned into pEGFP-Nl vector. The recombi-nant plasmid was identified by enzyme digestion and DNA sequencing. Then GFP and GFP-PTEN were respectively transfected into the osteosaocoma cell line( MG63 )with Lipofectamine 2000. The mRNA and protein expression level of PTEN gene in each cell group was detected by fluorescent quantitative RT-PCR and Western blot. Results Re-combinant vector of GFP-PTEN was constructed successfully. After transfection with GFP-PTEN, the mRNA and protein expression level of PTEN rose in MG63 cells. There were significant differences between transfected group and control group. Conclusion The eukaryotic expression vector of PTEN gene has been successfully constructed, which may provide a basis for further researches.
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