Construction and expression of enhanced green fluorescent protein and HGF co-expression vector

doi:10.13406/j.cnki.cyxb.2007.05.001

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Construction and expression of enhanced green fluorescent protein and HGF co-expression vector

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- Construction and expression of enhanced green fluorescent protein and HGF co-expression vector
- Journal of Chongqing Medical University Issue 5, Pages: 470-473(2007)
- 作者机构：
  
  重庆医科大学超声影像学研究所重庆医科大学附属第二医院超声科
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2007.05.001
  CLC： Q78
- Published：2007，
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[1]李兴升,王志刚,苏琳,任建丽,杨春江,郑元义,冉海涛,李攀.增强型绿色荧光蛋白与肝细胞生长因子共表达载体的构建及表达[J].重庆医科大学学报,2007(05):470-473.

Construction and expression of enhanced green fluorescent protein and HGF co-expression vector. [J]. Journal of Chongqing Medical University (5):470-473(2007)
[1]李兴升,王志刚,苏琳,任建丽,杨春江,郑元义,冉海涛,李攀.增强型绿色荧光蛋白与肝细胞生长因子共表达载体的构建及表达[J].重庆医科大学学报,2007(05):470-473. DOI： 10.13406/j.cnki.cyxb.2007.05.001.

Construction and expression of enhanced green fluorescent protein and HGF co-expression vector. [J]. Journal of Chongqing Medical University (5):470-473(2007) DOI： 10.13406/j.cnki.cyxb.2007.05.001.

摘要

目的:构建增强型绿色荧光蛋白与人肝细胞生长因子基因的真核共表达载体并鉴定其在真核细胞中的表达。方法:在HGF两端设计并合成分别带有Sac I和BamH I两酶切位点的一对引物

对pcDNA3.0-HGF载体进行PCR扩增

将扩增出的目的基因与pMD18-T载体相连

并转化大肠肝菌

进行菌落PCR、酶切分析和DNA测序鉴定重组克隆。用Sac I和BamH I将目的片段用双酶切切下后

纯化提取凝胶中的目的DNA片断

然后插入pIRES-EGFP相应的酶切位点

构建pIRES-EGFP/HGF真核表达载体并再进行酶切鉴定。将重组质粒用脂质体法转染至人脐带血管内皮细胞（HUVEC）中

荧光显微镜下观察EGFP在细胞内的表达及Western blotting检测HGF的表达情况。结果:重组克隆载体内的目的片段序列与GENE BANK上报道的HGF基因序列完全一致。pIRES-EGFP/HGF酶切鉴定结果与预期结果一致。荧光显微镜下观察可见转染后的细胞有荧光出现。Western blotting结果表明HGF基因得到了有效表达。结论:本实验成功构建EGFP和HGF真核共表达载体并可在真核细胞中有效表达。

Abstract

Objective:To construct EGFP and HGF co-expression vector and to detect its expression in cultured eukaryocyte. Methods:A pair of primers of HGF with Sac I and BamH I were designed and constructed

then PCR was performed.The PCR products were inserted into cloning vector pMDlg-T and transferred into JM109.The PCR of colony

restriction analysis

and DNA sequencing were performed to identify the correctness of the recombinant.The HGF gene fragment obtained from pMD18-T/HGF that was digested with Sac I and BamH I

and then inserted into pIRES2-EGFP that was cut with Sac I and BamH I.The recombinant pIRES2-EGFP/HGF was identified with restriction analysis.The expression plasmid pIRES2-EGFP/HGF was transfected into HUVEC mediated by liposome reagent

then the expression of EFGP in cell were ob- served by fluorescence microscopy and HGF protein was detected with Western blotting.Result:The sequence of the cloned DNA fragment was identical to HGF that was reported on Gene bank

and the HGF gene was inserted into eukaryotic expres- sion vector pIRES2-EGFP correctly.The recombinant expression plasmid was successfully transferred into HUVEC observed by fluorescent microscope and effective expression of HGF was also testified by Western blotting.Conclusion:The recombinant eukaryotic co-expression vector of EGFP and HGF was successfully constructed and effectively expressed in HUVEC.

关键词

肝细胞生长因子真核表达载体血管内皮细胞

Keywords

hepatocyte growth factoreukaryotic expression vectorvascular endothelial cell

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