Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (31): 5839-5842.doi: 10.3969/j.issn.1673-8225.2011.31.033

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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*

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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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