Construction and expression of the pIRES-enchanced green fluorescent protein reporter gene vector carrying BMP<sub>2</sub> Gene

ZOU Haibo; et al

doi:10.13406/j.cnki.cyxb.2004.04.014

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Construction and expression of the pIRES-enchanced green fluorescent protein reporter gene vector carrying BMP₂ Gene

更新时间：2024-01-17

- Construction and expression of the pIRES-enchanced green fluorescent protein reporter gene vector carrying BMP₂ Gene
- Journal of Chongqing Medical University Issue 4, Pages: 455-458(2004)
- 作者机构：
  
  1. 重庆医科大学临床学院骨科
  2. 重庆医科大学检验系,重庆,400016
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2004.04.014
  CLC： R346
- Published：2004，
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[1]邹海波,安洪,蒋电明,刘建伟,周亚莉.携带IRES的BMP_2绿色荧光蛋白表达载体的构建和表达[J].重庆医科大学学报,2004(04):455-458.

ZOU Haibo, et al. Construction and expression of the pIRES-enchanced green fluorescent protein reporter gene vector carrying BMP₂ Gene. [J]. Journal of Chongqing Medical University (4):455-458(2004)
[1]邹海波,安洪,蒋电明,刘建伟,周亚莉.携带IRES的BMP_2绿色荧光蛋白表达载体的构建和表达[J].重庆医科大学学报,2004(04):455-458. DOI： 10.13406/j.cnki.cyxb.2004.04.014.

ZOU Haibo, et al. Construction and expression of the pIRES-enchanced green fluorescent protein reporter gene vector carrying BMP₂ Gene. [J]. Journal of Chongqing Medical University (4):455-458(2004) DOI： 10.13406/j.cnki.cyxb.2004.04.014.

摘要

目的 :构建携带有BMP2 基因的pIRES2 绿色荧光蛋白表达载体

为BMP2 在体内外表达及蛋白定位提供标记。方法 :酶切 pCDAN3.1-BMP2 质粒

获得BMP2 cDNA片段并亚克隆至 pUC18载体上

随后用SalⅠ单酶切pUC18-BMP2

再次获得BMP2 cDNA

同时用SalⅠ将载体 pIRES2 -EGFP线性化后去磷酸化修饰。连接二者构建重组质粒。酶切筛选阳性克隆并鉴定插入的方向。重组质粒转染细胞

用激光共聚焦显微镜、RT -PCR及流式细胞仪检测基因在细胞内的定位、表达及转染效率 ;结果 :重组载体经酶切和测序证明构建正确

并在细胞中表达。结论 :成功构建了 pIRES2 -BMP2 -EGFP表达载体

为研究BMP2 的细胞内定位提供了一个重要而方便的工具

Abstract

Objective:To construct the pIRES2-enchanced green flurescent protein expression vector carrying BMP

gene in order to provide an ideal reporter gene for the expression and identify the location of portein in vitro and vivo.Methods:The BMP

cDNA sequence was excised

and subcloned on pUC18 vector.And then the BMP

sequence on the pUC18 plasmid was excised again and ligated into Sal Ⅰ site of pIRES2-EGFP treated by phosphorylase in sense and antisense direction.The correct recombinant plasmid was transfected into 293-T cells

the expression of BMP

was determined by FCM

LCM and RT-PCR.Results:The recombinant expression vector was verified correctly by enzyme digestion

sequence analysis.This vector was expressed in cells.Conclusion:A pIRES2 green fluorescent protein reporter gene vector containing BMP

has been constructed successfully

thus providing an important and convenient tool to study intracellular localization of BMP

关键词

骨形态发生蛋白绿色荧光蛋白内部核糖体进入位点真核表达

Keywords

BMP2green fluorescent proteinInternal ribosomes entry siteEukaryotic expression

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Construction and expression of the pIRES-enchanced green fluorescent protein reporter gene vector carrying BMP2 Gene

DOI：10.13406/j.cnki.cyxb.2004.04.014

摘要

Abstract

关键词

Keywords

references

Construction and expression of the pIRES-enchanced green fluorescent protein reporter gene vector carrying BMP₂ Gene