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Construction of tumor necrosis factor related apoptosis inducing ligand expression vector

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Author:
CAI Diao-long(Department of Surgery,Jia-zi People's Hospital of Lufeng(Lufeng 516538,China))
ZHU Guang-hui()
WENG Jie-feng()
XIA Jin-tang()
Journal Title:
CHINA MEDICINE
Issue:
Volume 3, Issue 04, 2008
DOI:
Key Word:
Tumor necrosis factor related apoptosis inducing ligand; Adeno-associated virus; Genetherapy

Abstract´╝Ü Objective To construct and identify recombined adeno associated virus encoding soluble tumornecrosis factor related apoptosis inducing ligand(sTRAIL)gene cDNA.Methods The shuttle plasmid pAAV-sTRAIL was first constructed.then transfected into HEK 293 cells by calcium phosphateprecipitation method.Recombinant adeno-associated viruS rAAV-sTRAIL Was produced by homologous recombination in 293 cells.Monoclonal rAAV-sTRAIL Was screened by plaque assay;virus identity was confirmed by PCR;purified virus stockswere prepared by CsCl gradients banding;virus particle titers were determined by OD260 measurements andfunctional titers in plaque forming units were determined by endpoint dilution infection on 293 cells. Western blotwas employed to detect the expression of the rAAV-sTRAIL in 293 cells.Results rAAV-sTRAIL was constructedand verified with hish particles titers and good purification.Furthermore,western blot showed that rAAV-sTRAILexpressed sTRAIL proteins in 293 cells.Conclusion The development of rAAV-sTRAIL provided an effective geneexpression vector tool for studv of the anti-tumor effect and for the clinical application of TRAIL cell.

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