| VEGF165真核表达载体的构建与体外表达 |
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| 引用本文： | 於丽明,王佐林. VEGF165真核表达载体的构建与体外表达[J]. 口腔颌面外科杂志, 2009, 19(1): 9-14 |
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| 作者姓名： | 於丽明 王佐林 |
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| 作者单位： | 同济大学附属口腔医院口腔颌面外科,上海,200072 |
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| 摘 要： | 目的:体外构建血管内皮生长因子(vascular endothelial growth factor,VEGF)165的荧光真核表达载体,并检测其在293T细胞中的表达。方法:应用RT-PCR方法从人单核细胞白血病细胞株THP-1中分离、扩增目的片段,单酶切克隆到真核表达载体pEGFP-C1中,构成重组质粒pEGFP-VEGF165,测序验证。通过脂质体介导质粒瞬时转染293T细胞,RT-PCR和免疫细胞化学检测VEGF165的表达。结果:通过RT-PCR获得576bp的cDNA片段,测序结果与基因库(Genbank)中序列完全一致。转染后经RT-PCR和免疫细胞化学检测证实转染重组质粒组有VEGF165蛋白表达,而转染空质粒组及未转染组没有检测到VEGF的表达。结论:本研究构建了人VEGF165荧光真核表达载体pEGFP-VEGF165,并能成功地在293T细胞中表达。
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| 关 键 词： | 血管内皮生长因子 基因克隆 基因转染 |
Construction of Recombinant Plasmid pEGFP-VEGF165 in vitro and Expression in 293T Cells |
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| YU Li-ming,WANG Zuo-lin. Construction of Recombinant Plasmid pEGFP-VEGF165 in vitro and Expression in 293T Cells[J]. Chinese Journal of Oral and Maxillofacial Surgery, 2009, 19(1): 9-14 |
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| Authors: | YU Li-ming WANG Zuo-lin |
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| Affiliation: | Department of Oral and Maxillofacial Surgery;Hospital of Stomatology;Tongji University;Shanghai 200072;China |
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| Abstract: | Objective:To construct fluorescent recombinant plasmid pEGFP-VEGF165 in vitro and to evaluate its expression in 293T cells.Methods:The target sequence of human VEGF165 was obtained and amplified from THP-1 cells by RT-PCR.The cDNA segment was cloned into a eukaryote plasmid pEGFP-C1 after restrictive endonucleases digestion.The pEGFP-VEGF165 was checked and verified by DNA sequence analysis.293T cells were transiently transfected with pEGFP-VEGF165 by lipofectamine 2000 in vitro.RT-PCR and immunocytochemica... |
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| Keywords: | vascular endothelial growth factor 165（VEGF165） gene clone gene transfection |
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