期刊文献+

含绿色荧光蛋白和PLC_Z的逆转录病毒载体的构建及其在SHG44细胞中的表达

Construction of retroviral vector containing green fluorescent protein and PLC_Z and its expression in SHG44 glioma cells
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摘要 目的 构建含有 PL C- γ1分子 Z区 (PL CZ)和绿色荧光蛋白基因的双顺反子逆转录病毒载体 ,并将其导入 SHG44胶质瘤细胞中 ,以探索 PL C-γ1分子对胶质瘤细胞侵袭作用的影响 .方法  PCR法扩增 PL Cz基因 ,测序后克隆入p IRES- EGFP荧光蛋白表达载体中 ,酶切鉴定重组体 .将PL Cz- IRES- EGFP片段克隆入逆转录病毒载体 p L XSN的相应位点 ,构建含有 PL Cz和 EGFP基因的逆转录病毒载体 ,脂质体介导下转染包装细胞 PA317,G418筛选并检测病毒滴度后挑选细胞克隆 .病毒上清感染人脑胶质瘤 SHG44细胞 ,G418筛选获得稳定转染细胞 ,PCR检测外源基因的整合 ,Northern blot检测外源基因的转录 ,Western blot和荧光显微镜分别检测 PL Cz分子和 EGFP的表达 .结果 PCR扩增得到大小约 6 5 0 bp的特异性条带 ,序列测定结果与发表序列一致 .构建得到含有 PL Cz和 EGFP基因的双顺反子逆转录病毒载体 ,转染人脑胶质瘤细胞 SHG44 ,得到稳定转染的细胞株 SHG44 - PIE.PCR扩增显示目的片段已整合入细胞基因组 .Northern blot分析显示为单一的转录本 .Western blot可见大小约 5 0× 10 3的特异性条带 ,荧光显微镜检测显示EGFP获得良好表达 .结论 成功构建含有 PL Cz和绿色荧光蛋白基因的逆转录病毒载体 ,并获得稳定转染该载? AIM The retroviral vector containing green fluorescent protein and Z region gene of Phospholipase C γ1(PLC Z) was constructed and transduced into SHG44 glioma cells to probe for the effects of phospholipase C γ1 on the invasion of glioma. METHODS PLCz gene was amplified with PCR method and then cloned into the pIRES EGFP expression vector after its sequence was determined. The recombinant vector was identified by enzymatic digestion. The fragment encoding PLCz IRES EGFP was cloned into appropriate site of pLXSN retroviral vector to construct the retroviral vector containing EGFP and PLCz gene. The constructed retroviral vector was transfected into PA317 cells under mediation of LipofectAMINE. Positive clones were selected by G418 and their virus titer was examined. TheSHG44 cells were infected by virus supernatant and stably transfected cell clones were obtained by G418 selection. The integration of exotic gene was examined by PCR method and the transcription of gene was determined by northern blot. The expression of PLCz and EGFP were respectively determined by western blot and fluorescence microscope. RESULTS Special strap about 650 bp was obtained and its sequence was consistent with sequence that had been published. The retroviral vector containing PLCz and EGFP gene was successfully constructed. After this vector was transfected into SHG44 glioma cells, the stable cell line (SHG44 PIE) was obtained. The integration of objective fragment was confirmed by PCR and northern blot assay revealed a single LTR promoted transcript. The special strap about 50×10 3 was determined by western blot and the expression of EGFP was examined by fluorescent microscope. CONCLUSION Retroviral vector containing PLCz and EGFP gene is successfully constructed and the SHG44 cell line stably transfected with this gene is obtained.
出处 《第四军医大学学报》 北大核心 2001年第23期2125-2129,共5页 Journal of the Fourth Military Medical University
基金 国家自然科学基金资助项目 ( 39970 75 2 )
关键词 绿色荧光蛋白 磷脂酶C 逆转录病毒载体 脑胶质瘤 green fluorescent protein phospholipase C retroviral vector glioma
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参考文献5

  • 1Lu F,第四军医大学学报,2001年,22卷,6期,522页
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