Construction and prokaryotic expression of fusion gene IL3-PE38KDEL

CHEN Yu; et al

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Construction and prokaryotic expression of fusion gene IL3-PE38KDEL

更新时间：2024-01-17

- Construction and prokaryotic expression of fusion gene IL3-PE38KDEL
- Journal of Chongqing Medical University Vol. 34, Issue 8, Pages: 1028-1031(2009)
- 作者机构：
  
  1. 重庆医科大学附属第二医院血液内科
  2. 重庆医科大学检验系重点实验室
- 作者简介：
- 基金信息：
- DOI：
  CLC： Q78
- Published：2009，
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[1]陈妤,娄世锋,邓建川.IL3-PE38KDEL融合基因的构建及原核表达[J].重庆医科大学学报,2009,34(08):1028-1031.

CHEN Yu, et al. Construction and prokaryotic expression of fusion gene IL3-PE38KDEL. [J]. Journal of Chongqing Medical University 34(8):1028-1031(2009)
[1]陈妤,娄世锋,邓建川.IL3-PE38KDEL融合基因的构建及原核表达[J].重庆医科大学学报,2009,34(08):1028-1031. DOI：

CHEN Yu, et al. Construction and prokaryotic expression of fusion gene IL3-PE38KDEL. [J]. Journal of Chongqing Medical University 34(8):1028-1031(2009) DOI：

摘要

目的:构建原核表达载体PQE30-IL3-Linker-PE38KDEL

并诱导和鉴定其蛋白表达。方法:用PCR方法扩增所需要的目的片段IL3及PE38KDEL再通过酶切和连接的方法定向克隆到载体PQE30-Linker中得到融合基因PQE30-IL3-Linker-PE38KDEL。重组载体经酶切

菌落PCR鉴定

DNA序列分析插入片段完全正确

转化感受态大肠杆菌SG13009

经IPTG诱导

SDS-PAGE分析分子量大小

Western blot鉴定。结果:经限制性内切酶酶切鉴定

菌落PCR及DNA序列分析表明重组表达载体PQE30-IL3-Linker-PE38KDEL构建成功

IPTG诱导后得到了与预计分子量相符的目的蛋白。经Western blot鉴定在分子量57kD处有明显特异性条带

说明目的蛋白正确表达。结论:成功构建融合蛋白IL3-PE38KDEL

为后续的蛋白质纯化及功能研究奠定基础。

Abstract

Objective:To construct a prokaryotic expression plasmid of PQE30-IL3-Linker-PE38KDEL and identify its recombinant protein expression.Methods:The IL3 and PE38KDEL gene were amplified by polymerase chain reaction(PCR) and cloned into the prokaryotic expression plasmid PQE30-Linker constructed after being sequenced.The recombinant vector confirmed by restriction endonucleases digestion

coenobium PCR

and DNA sequence analysis was transformed into E.coli SG13009.The expression of the protein was induced by IPTG.Relative molecular weight of the expression product was detected by SDS-PAGE.Finally

the fusion protein was examined by Western blot.Results:The results of restriction endonuclease digestion

coenobium PCR and DNA sequence analysis showed that the prokaryotic expression vector PQE30-IL3-Linker-PE38KDEL was constructed successfully.With induction of IPTG

the relative molecular weight of the expression product was identical to the expected value.The expressed 6×His-IL3-PE38KDEL fusion protein were identified at relative molecular mass of 57KD by Western blot with anti-His monoclonal antibody

showing the fusion protein expressed correctly.Conclusion:The fusion protein IL3-PE38KDEL is successfully constructed

which lays a solid foundation for the further research of protein purification and function.

关键词

融合基因白介素3PE38KDEL原核表达PQE30

Keywords

Fusion geneInterleukin 3(IL3)PE38KDELprokaryotic expressionPQE30

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