中国现代医学杂志2017，Vol.27Issue(16)：12-17,6.DOI:10.3969/j.issn.1005-8982.2017.16.003
小鼠11β-HSD1基因真核表达载体的构建及应用
Construction and role of mouse PLJM1-11β-HSD1-GFP plasmid
辛婧 1叶磊 2杨可 1沈亚非 1邓飞1
作者信息
- 1. 河南省漯河市中心医院(漯河市医学高等专科学校第一附属医院)内分泌科,河南 漯河 462000
- 2. 河南省漯河市召陵区人民医院 神经内科,河南 漯河 462000
- 折叠
摘要
Abstract
Objective To construct a lentiviral vector of PLJM1-11β-HSD1-GFP and establish a stable cell line of 3T3-L1 with high expression of mouse 11β-HSD1 gene, and lay the foundation for the research of 11β-HSD1 gene. Methods Open reading frame (ORF) fragment of mouse 11β-HSD1 gene was amplified from mouse liver tissues by RT-PCR, then inserted into the PLJM1-NRG1-GFP vector and transformed into competent DH5α strain of Escherichia coil. The plasmid was extracted and used for sequencing. The successfully-sequenced plasmid PLJM1-11β-HSD1-GFP and the packaging plasmid were transfected to 293T cell line to produce recombinant lentivirus. 3T3-L1 cell line was transfected by recombinant virus and the stable cell line was isolated by selecting the single clone with GFP, and high expression of 11β-HSD1 was identified by Western blot. Results The eukaryotic expression vector of PLJM1-11β-HSD1-GFP was constructed and confirmed by DNA sequencing. The lentivirus vector of 11β-HSD1 named PLJM1-11β-HSD1-GFP was successfully constructed and the virus was packaged in 293T cells. 3T3-L1 cells were transfected by recombinant virus and the stable cell line was selected by green fluorescence efficiency, which showed that the lentivirus vector transfection rate was over 90%. A dramatically elevated protein level of 11β-HSDl was expressed in the positive clones of 3T3-L1 cells compared with the control group. Conclusions Mouse PLJM1-11β-HSD1-GFP plasmid has been successfully constructed. The 3T3-L1 cells transfected by PLJM1-11β-HSD1-GFP could effectively elevate the expression of 11β-HSD1 gene, and can be used in the functional researches of mouse gene, especially those related to obesity.关键词
11β-HSD1基因/真核表达载体/前体脂肪细胞/肥胖Key words
11β-HSD1 gene/eukaryotic expression vector/3T3-L1 cell/obesity分类
生物科学引用本文复制引用
辛婧,叶磊,杨可,沈亚非,邓飞..小鼠11β-HSD1基因真核表达载体的构建及应用[J].中国现代医学杂志,2017,27(16):12-17,6.
