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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2009, Vol. 3 Issue (3) : 297-302     DOI: 10.1007/s11684-009-0058-x
Research articles |
Construction of eukaryotic expression vector of human arresten gene and its secreted expression in HEK 293 cells
Wei LI PhD 1, Siming GUAN MM 1, Zifang SONG PhD 2, Qichang ZHENG PhD 2, Jun XIONG PhD 2, Dan SHANG PhD 2, Xiaogang SHU PhD 2,
1.Department of Gerontology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 2.Department of General Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China;
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Abstract  The eukaryotic expression vector of human arresten gene was constructed and its secretive expression human embryonic kidney (HEK 293) cells was detected. Human arresten gene was amplified from recombinant plasmid pGEM-Arr by polymerase chain reaction (PCR), and then digested with restriction endonucleases BamH I and EcoR I. The target fragment was inserted into the BamH I and EcoR I restriction sites of eukaryotic expression vector pSecTag2 to construct pST-AT. Restriction analysis and DNA sequencing indicated that the arresten gene was successfully inserted into pSecTag2. The recombinant plasmid was subsequently transfected into HEK 293 cells with LipofectAMINETM2000 Reagent, and the expression of the target gene was detected. RT-PCR revealed that the mRNA of the target gene was transcribed in the transfected HEK 293 cells. Western Blot analysis verified that the recombinant protein in supernatants was correct. The supernatants of transfected cells were prepared, and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was carried out to assess their effect on the proliferation of human umbilical vein endothelial cells, which showed that the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells in vitro. These results provided a solid foundation to explore the usage of arresten in tumor anti-angiogenic gene therapy.
Keywords angiogenesis inhibitor      arresten      eukaryotic expression      HEK 293 cells      endothelial cells      
Issue Date: 05 September 2009
URL:     OR
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