Objective:To construct lentiviral vector of recombinant BTLA gene and investigate the effects on proliferation of mice splenic T lymphocytes after overexpression of BTLA gene mediated by lentiviral vector. Methods:The BTLA gene was cloned into the lentiviral expression vector (pWPTS-GFR),which was verified by reverse transcription and PCR. The lentiviral vector of recombinant BTLA gene was co-transfected into 293T cells. The morphological changes of 293T cells infected by lentiviral granules were observed by fluorescence microscope and lentivirus titer was tested by the method of TCID50. Results:pWPTS-mBTLA lentivrial vector at a titer of 1.3×108 pfu /mL was successfully constructed. Overexpression of BTLA gene in mice splenic T lymphocytes significantly suppressed the activation and proliferation of mice splenic T lymphocytes on day 4 and 8(P < 0.05). Conclusion:Outcomes of the inhibiting effect in activation and proliferation of mice splenic T lymphocytes induced by overexpression of BTLA gene indicated the successful construction of lentiviral vector of the recombinant BTLA gene.