稳定表达细胞内病原体抗性基因1的RAW264.7细胞克隆的建立
Establishment of RAW264.7 cell clones stably expressing intracellular pathogen resistance 1
王瑜伟 1朱道银 1李娜1
作者信息
- 1. 重庆医科大学基础医学院免疫学教研室,重庆400016
- 折叠
摘要
目的:构建携带细胞内病原体抗性基因1(Ipr1),以绿色荧光蛋白(GFP)为报告基因的重组真核表达载体,并导入鼠巨噬细胞RAW264.7中表达.方法:Kpn Ⅰ和BamH Ⅰ双酶切重组质粒pET32a(+)-Ipr1及真核表达载体pEGFP-C1,Ipr1基因定向克隆入pEGFP-C1载体,构建重组质粒载体pEGFP-C1一Ipr1.脂质法转染体培养的RAW264.7细胞,在活细胞状态下用荧光显微镜直接观察pEGFP-C1-Ipr1融合蛋白在细胞中的表达;PCR检测Ipr1基因转录水平的表达;West-ern Blot方法验证Ipr1蛋白水平的表达.结果:酶切鉴定获得一条约4700 bp卒载体条带及一条约1338 bp目的片段,证明pEGFP-C1-Ipr1真核表达载体构建成功.pEGFP-C1-Ipr1转染RAW264.7细胞后,Western Blot可以检测到约Mr 77×103的融合蛋白在RAW264.7细胞中表达,荧光显微镜下可观察纠转染细胞中有绿色荧光蛋白表达.结论:成功构建真核绿色荧光蛋白表达载体pEGFP-C1-Ipr1,获得稳定的RAW264.7-Ipr1细胞克隆可表达Ipr1,为进一步研究lpr1的功能奠定了基础.
Abstract
AIM: To construct pEGFP-C1-Ipr1 (intracellular pathogen resistance 1) eukaryotic expression vector using green fluorescence protein as reporter gene and to express it in RAW264. 7 cells. METHODS: The pET32a ( + )-Ipr1 and pECFP-C1 vectors were digested by Kpn Ⅰ /BamH Ⅰ and purified by gel extraction. The Ipr1 genes were inserted into pEGFP-C1 vector. The RAW264.7 cells were transfected with the constructs by LipofectaminTM 2000. The EGFP-Ipr1 fused protein was visualized directly under fluorescence microscope and the expression of Ipr1 was detected by Western blot and PCR at protein and gene levels respectively. RESULTS: The fragments with size of 4700 bp and 1338 bp were obtained by enzyme digestion and a fusion protein about 77 ku was observed by Western Blot. Restriction enzyme digestion and sequence analysis showed that the recom-binant vector pEGFP-C1-Ipr1 was constructed successfully, which was stably expressed in eukaryotic cells. Ipr1 gene and protein were detected in the transfected RAW264. 7 cells and the expressed EGFP was observed under fluorescence microscope. CONCLUSION: The eukaryotic expression vector named pEGFP-C1-Ipr1 is successfully constructed and the RAW264. 7 cell clone stably expressing Ipr1 is obtained, which provides a good basis for further research on the function of Ipr1 against tuberculosis.
关键词
细胞内病原体抗性基因1/转染/脂质体/细胞克隆Key words
Ipr1/liposomes/transfection/cell cloning引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据