中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (31): 5839-5842.doi: 10.3969/j.issn.1673-8225.2011.31.033

• 器官移植动物模型 organ transplantation and animal model • 上一篇    下一篇

真核表达载体pIRES2-EGFP-hFasL的构建及鉴定

邱明链1，方航荣2，陈丽红3，刘景丰4   

  1. 福建医科大学附属第一医院，1胸外科，4肝病中心，福建省福州市 350005；2西安医学院附属医院病理科，陕西省西安市 710077；3福建医科大学基础医学院病理系，福建省福州市 350004
  • 收稿日期:2011-04-21 修回日期:2011-05-28 出版日期:2011-07-30 发布日期:2011-07-30
  • 作者简介:邱明链☆，男，1977年生，福建省仙游县人，汉族，2008年福建医科大学毕业，博士，主治医师，主要从事移植研究。 qml-817@163. com
  • 基金资助:

    福建省自然科学基金资助项目(2009J05061)。

Construction and identification of eukaryotic expression vector pIRES2-EGFP-hFasL

Qiu Ming-lian1, Fang Hang-rong2, Chen Li-hong3, Liu Jing-feng4   

  1. 1Department of Thoracic Surgery, 4Hepatopathy Center, First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian Province, China; 2Department of Pathology, Affiliated Hospital of Xi’an Medical College, Xi’an 710077, Shaanxi Province, China; 3Department of Pathology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350004, Fujian Province, China
  • Received:2011-04-21 Revised:2011-05-28 Online:2011-07-30 Published:2011-07-30
  • About author:Qiu Ming-lian☆, Doctor, Attending physician, Department of Thoracic Surgery, First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian Province, China qml-817@163.com
  • Supported by:

    the Natural Science Foundation of Fujian Province, No. 2009J05061*

PDF

329

被引次数

1

摘要：

背景：FasL通过与靶细胞上Fas结合诱导程序性细胞死亡，可维持机体内稳态，诱导同种异基因移植免疫耐受，促进肿瘤细胞凋亡。
目的：构建带有增强型绿色荧光蛋白报告基因EGFP及目的基因hFasL的真核表达载体pIRES2-EGFP-hFasL。
方法：通过实时聚合酶链反应RT-PCR方法从人外周血淋巴细胞中扩增出hFasL基因，与真核空载体 plRES2-EGFP一起，经XhoⅠ和EcoRⅠ双酶切，T4 DNA连接酶连接，从而构建pIRES2-EGFP-hFasL。用紫外分光光度计测定质粒浓度及纯度，经酶切、PCR技术、基因测序等方法进行鉴定。
结果与结论：扩增出的hFasL条带大小约846 bp，构建的plRES2-EGFP-hFasL经酶切后在846 bp和2 000 bp处有特异性条带，DNA测序证实hFasL与Genebank上的序列完全一致。提示成功构建了含有hFasL及EGFP的真核表达载体plRES2-EGFP-hFasL。

关键词: 基因, FasL, 增强型绿色荧光蛋白, 载体, 基因重组

Abstract:

BACKGROUND: FasL induced target cells to programmed cell death by binding with Fas, which was critically important to steady-state mechanism for the body, immune tolerance and tumor apoptosis mechanisms.
OBJECTIVE: To construct the eukaryotic expression vector pIRES2-EGFP-hFasL containing enhanced green fluorescent protein report gene (EGFP) and target gene hFasL.
METHODS: FasL gene was amplified from human peripheral blood lymphocytes by using real time polymerase chain reaction (RT-PCR), then plRES2-EGFP-hFasL plasmid was constructed through the XhoⅠand EcoRⅠdouble digestion and T4 DNA ligase conjunction. The plasmid concentration and purity were detected by ultraviolet spectrophotometey and identified by endonuclease digestion, PCR and sequencing.
RESULTS AND CONCLUSION: The amplified hFasL gene was about 846 bp in length. After digestion of recombinant plasmid plRES2-EGFP-hFasL by restrictive enzymes, specific products with a size of 846 bp and 2000 bp were obtained. DNA sequencing indicates 100% coincidence between hFasL sequences of plRES2-EGFP-hFasL and Genebank. These finding suggest that the eukaryotic expression vector plRES2-EGFP-hFasL containing EGFP and hFasL genes was successfully constructed.

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