Eukaryotic expression of cis-inserted double copies of DuIFN-γ gene

HUANG Ai-long; et al

doi:10.13406/j.cnki.cyxb.2001.04.003

您当前的位置：

首页 >

文章列表页 >

Eukaryotic expression of cis-inserted double copies of DuIFN-γ gene

更新时间：2024-01-17

- Eukaryotic expression of cis-inserted double copies of DuIFN-γ gene
- Journal of Chongqing Medical University Issue 4, Pages: 368-371(2001)
- 作者机构：
  
  1. 重庆医科大学病毒性肝炎研究所
  2. 澳大利亚阿得雷德大学微生物与免疫学系
  4. 南澳州,5005
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2001.04.003
  CLC： R346
- Published：2001，
扫描看全文
[1]黄爱龙,单幼兰,Allison Jilbert,Ieva Kotlarski.顺式串联双拷贝DuIFN-γ基因的真核表达[J].重庆医科大学学报,2001(04):368-371.

HUANG Ai-long, et al. Eukaryotic expression of cis-inserted double copies of DuIFN-γ gene. [J]. Journal of Chongqing Medical University (4):368-371(2001)
[1]黄爱龙,单幼兰,Allison Jilbert,Ieva Kotlarski.顺式串联双拷贝DuIFN-γ基因的真核表达[J].重庆医科大学学报,2001(04):368-371. DOI： 10.13406/j.cnki.cyxb.2001.04.003.

HUANG Ai-long, et al. Eukaryotic expression of cis-inserted double copies of DuIFN-γ gene. [J]. Journal of Chongqing Medical University (4):368-371(2001) DOI： 10.13406/j.cnki.cyxb.2001.04.003.

摘要

目的：以鸭γ-干扰素（DuIFN-γ）基因为目的基因，研究同一启动子调控下，不同拷贝数的目的基因的表达状况。方法：采用基因重组技术构建含DuIFN-γcDNA单拷贝和双拷贝重组质粒并转入真核细胞COS-7中进行表达。DuIFN-γ活性采用Griess试剂检测。结果：双拷贝重组质粒和单拷贝重组质粒50%最大活性稀释度分别为1∶320和1∶160。双拷贝重组质粒转染上清稀释10240倍后，仍保留DuIFN-γ活性，而单拷贝重组质粒转染上清经1280倍稀释后，即丧失活性。结论：在同一启动子作用下，顺式插入双拷贝目的基因可明显增强目的基因在真核细胞中的表达量。

Abstract

Objective:In this study

duck interferon-gamma(DuIFN-γ) gene has been used as target gene to investigate the effect of copy number of cloned foreign gene on the expression of target protein under control of a single promoter .Methods:The 1.4kb size of DuIFN-γ cDNA was cleaved from plasmid pGEM-DuIFN-γ with EcoRI and subcloned into the EcoRI site of pcDNA1.1/Amp vector.The copy number and orientation of insert in recombinant plasmids were characterised by restriction enzyme analysis and transfected into COS-7 cells for expression with FuGENE6 transfection reagent.The biological activity of DuIFN-γ in cultured supernatant was assayed with Griess reagent for its capacity of activating chicken macrophage cell line HD11 to secrete nitric oxide. The recombinant plasmids with one or two copies of forward inserts were identified with restriction enzyme analysis.Results: The results of DuIFN-γ biological assay showed that 50% of maximal DuIFN-γ activity was reserved after 1∶160 dilution of cultured supernatant transfected by recombinant plasmid with single copy of DuIFN-γ cDNA

the cultured supernatant of transfected COS-7 cells remained half of the maximal activity of DuIFN-γ following 1∶320 dilution.In addition

the DuIFN-γ was still active after 10240 fold dilution for plasmid wth double copies of DuIFN-γ genes

but inactive after 1280 fold dilution for plasmid with single copy of DuIFN-γ gene.Conclusion:By sharing the same promoter

the level of target protein (DuIFN-γ) expressed by recombinant plasmid with two copies of forward insert was obviously enhanced in eukaryotic cells.

关键词

真核表达γ-干扰素基因重组基因调控

Keywords

Eukaryotic expressionInterferon-gammaGene recombinationGene regulation

references

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

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

Construction of eukaryotic expression vector of open reading frame of unknown KH gene and its effect on proliferation of K562 cell

Construction of pcDNA3.1(+)/hFasL and its expression in COS-7 cells

Construction and verification of eukaryotic expression vector for mouse Hsp84

Establishment of high efficient tranfection and expression system for phPPARy 1-1RES2-EGFP recombinant plasmid in vitro

Related Author

周亚莉

刘建伟

蒋电明

et al

ZOU Haibo

刘小珊

HE Yujuan

et al

Related Institution

重庆医科大学检验系

Department of Orthopaedic Surgery,College of Clinical Medicine,Chongqing Medical University

永川

重庆医科大学基础医学院临床生化教研室

重庆市永川市人民医院

⁰