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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?
Objective To construct a bicistronic retroviral vector containing PLAC-γ1 Z-region (PL CZ) and green fluorescent protein gene and introduce it into SHG44 glioma cells to explore the role of PL C-γ1 molecule in Effect of Tumor Cell Invasion. METHODS: PCR was used to amplify the PL Cz gene, which was cloned into the pIRES-EGFP fluorescent protein expression vector after sequencing. The recombinants were digested and identified. The PL Cz-IRES-EGFP fragment was cloned into a retroviral vector. At the corresponding sites of p L XSN, a retroviral vector containing PL Cz and EGFP genes was constructed, and transfected with packaging cells PA317 and G418 were selected for liposome-mediated transfection and G418 selection and cell clones were selected. The virus supernatant was infected. In glioma SHG44 cells, stable transfected cells were screened with G418, the integration of foreign genes was detected by PCR, the transcription of foreign genes was detected by Northern blot, and the expression of PL Cz molecules and EGFP were detected by Western blot and fluorescence microscopy, respectively. A specific band of approximately 650 bp in size was obtained and the sequence was identical to the published sequence. A bicistronic retroviral vector containing the PL Cz and EGFP genes was constructed and transfected into human brain gels. The tumor cell SHG44 was obtained stably transfected cell line SHG44 - PIE. PCR amplification showed that the target fragment had been integrated into the cell genome. Northern blot analysis showed a single transcript. Western blot showed a specific size of about 50×10 3 The band was detected by fluorescence microscopy to show that EGFP was well expressed. Conclusion The retroviral vector containing PL Cz and green fluorescent protein gene was successfully constructed, and stable transfection of the vector was obtained.