| 摘要: |
| 目的:构建胰腺癌新基因S100P与绿色荧光蛋白(GFP)融合基因慢病毒载体。方法:实时定量聚合酶链反应(RT-PCR) 方法扩增获得S100P的全外显子片段,使之克隆到带GFP荧光报告基因慢病毒表达载体质粒中,慢病毒包装质粒和穿梭质粒共转染293T细胞,包装成功后收集上清,浓缩,鉴定。取浓缩纯化后的病毒上清感染293T细胞和宿主胰腺癌细胞,荧光显微镜观察293T细胞的荧光表达,RT-PCR鉴定胰腺癌细胞中S100P的表达水平。结果:电泳鉴定结果与目的基因表达条带完全吻合,克隆测序结果与NCBI收录的S100P基因序列完全一致。重组慢病毒质粒可高效转染293T细胞,荧光显微镜下可观察到大量绿色荧光。共转染后293T细胞上清可高效感染293T细胞,感染后宿主细胞中S100P高表达。 结论:成功构建了S100P与GFP融合基因慢病毒表达载体,为进一步研究S100P基因的相关功能提供了适合的稳定转染载体。 |
| 关键词: 慢病毒 S100P 绿色荧光蛋白 |
| DOI：10.3724/SP.J.1008.2008.01230 |
| 投稿时间：2008-01-30修订日期：2008-04-11 |
| 基金项目:上海市浦江人才计划资助(05PJ14006). |
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| Construction of lentivirus carrying novel human pancreatic cancer gene S100P and green fluorescent protein gene |
| LI Gang1,JIN Gang1*,ZHOU Xu-yu1,HU Xian-gui1,WANG Jun2,LI Tie-jun2 |
| (1. Department of General Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China*2. Department of Pharmacology, School of Pharmacy, Second Military Medical University, Shanghai 200433) |
| Abstract: |
| Objective:To construct lentivirus carrying both novel human pancreatic cancer gene S100P and green fluorescent protein (GFP) gene.Methods: The fragments containing all the exons of S100P were amplified by RT-PCR and were cloned into the lentivirus vectors labeled with GFP. The lentivirus was packaged and was used to transfect 293T cells together with pShuttle. The supernatant of virus-producing cells was harvested, concentrated, identified, and was used to infect 293T cells and pancreatic cancer cells. Fluorescent microscopy was used to observe the fluorescence in the 293T cells; and real time-PCR was used to examine the relative contents of S100P in pancreatic cancer cells.Results: Electrophoresis showed that the sequence of the RT-PCR product was consistent with the data of NCBI by DNA sequencing analysis. The lentivirus effectively transfected 293T cells. Strong green fluorescence was observed by fluorescent microscopy. The supernatant of lentivirus-transfected 293T cells effectively infected 293T cells and the relative content of S100P in the transfected pancreatic cancer cells was higher than that of control group.Conclusion: The lentivirus vector containing S100P-GFP recombinant gene have been successfully constructed, which provides a basis for further study of S100P function. |
| Key words: Lentivirus S100P green fluorescent protein |
