Construction and identification of the recombination Lentiviral vector with human IDO gene

Objective: To construct the recombination lentiviral expressing vector with human IDO gene.Methods: The full-length coding region fragment of IDO was amplified by primer designed by the cDNA library including IDO gene using polymerase chain reaction(PCR).The target gene was directly inserted into the pGC-FU lentiviral vector,which were transformed into the Bacterium coli DH5α cells.The positive clone was identified by PCR and sequencing.The recombinant lentiviral vector and packaging plasmids phelper 1.0 and pHelper 2.0 were cotransfected into 293T cells.The expression of green fluorescent protein(GFP) was observed to evaluate gene delivery efficiency by fluorescence microscope.The levels of fusion protein IDO-GFP expression were detected by Western Blot,and the titers of lentiviral were determined by Real-time PCR.Results: The encoding sequences of IDO were successfully obtained by the analysis of PCR,the recombination lentiviral vector with human IDO gene was successfully constructed,and transfected into 293T cells to express IDO.The titer of lentivirus was 2×10⁸ TU/ml.Conclusions: The recombinant lentiviral vector with high titer is successfully constructed,which can provide the basis of the future experiment.