Construction of eukaryotic expression vector encoding antimicrobial peptides PR-39 and its expression in 293T cell

ZHU Yong-jun; et al

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Construction of eukaryotic expression vector encoding antimicrobial peptides PR-39 and its expression in 293T cell

更新时间：2024-01-17

- Construction of eukaryotic expression vector encoding antimicrobial peptides PR-39 and its expression in 293T cell
- Journal of Chongqing Medical University Vol. 34, Issue 12, Pages: 1643-1645(2009)
- 作者机构：
  
  1. 重庆医科大学附属第一医院骨科
  2. 重庆医科大学医学检验生物化学教研室,临床检验诊断学省部共建教育部重点实验室
- 作者简介：
- 基金信息：
- DOI：
  CLC： Q78
- Published：2009，
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[1]朱勇军,翁玄,张健,文阳安.抗菌肽PR-39真核表达载体的构建及表达检测[J].重庆医科大学学报,2009,34(12):1643-1645.

ZHU Yong-jun, et al. Construction of eukaryotic expression vector encoding antimicrobial peptides PR-39 and its expression in 293T cell. [J]. Journal of Chongqing Medical University 34(12):1643-1645(2009)
[1]朱勇军,翁玄,张健,文阳安.抗菌肽PR-39真核表达载体的构建及表达检测[J].重庆医科大学学报,2009,34(12):1643-1645. DOI：

ZHU Yong-jun, et al. Construction of eukaryotic expression vector encoding antimicrobial peptides PR-39 and its expression in 293T cell. [J]. Journal of Chongqing Medical University 34(12):1643-1645(2009) DOI：

摘要

目的:构建富含脯氨酸精氨酸的39位氨基酸抗菌肽（Proline-arginine rich 39-amino acid peptide

PR-39）的真核表达载体

并检测它在293T细胞中的表达情况。方法:以含有PR-39核心编码区（Core coding regions

CDS）序列的质粒pBluescriptⅡSK-PR-39为模板

PCR扩增PR-39CDS序列并测序。扩增片段插入真核表达质粒pGC-FU中

构建包含PR-39基因的重组表达质粒pGC-FU-PR-39。阳性克隆用限制性内切酶酶切及测序鉴定后用脂质体2000转染293T细胞

荧光显像观察其转染效率

Western blot检测目的蛋白表达情况。结果:成功扩增出PR-39基因

通过酶切测序鉴定证明成功构建了包含PR-39的真核表达载体pGC-FU-PR-39

转化293T细胞24h后观察到荧光

Western blot检测到目的蛋白。结论:本实验成功构建PR-39真核表达载体

并能在293T细胞中表达PR-39-GFP融合蛋白

这为进一步研究PR-39的作用奠定了基础。

Abstract

Objective:To construct eukaryotic plasmid expressing PR-39 and test whether the plasmid can be expressed in the 293 T cell.Methods:The core coding regions(CDS) sequence of PR-39 was amplified by PCR from pBluescriptⅡSK-PR-39.Then the PR-39 gene was inserted into the eukaryotic expression plasmid pGC-FU.The resultant recombinant plasmid was confirmed by restriction enzyme cutting and sequencing

and then was designated as pGC-FU-PR-39.The recombinant plasmid pGC-FU-PR-39 was transfected into 293 T cell by Lipofectamine 2000.Transfected efficiency was observed by fluorographic unit.Western blot was used to certify whether the purpose protein was correctly expressed in 293 T cell.Results:The CDS sequence of PR-39 was correctly amplified.The recombinant plasmid was successfully constructed and could be correctly expressed in 293 T cell.Conclusion:We successfully constructed PR-39 eukaryotic expression plasmid and observed the expression of PR-39-GFP fusion protein.It lays a foundation for further studies of PR-39.

关键词

抗菌肽PR-39真核表达载体

Keywords

antimicrobial peptidePR-39Eukaryotic expression

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