The LAG3 plasmid and lentiviral vector including mCherry gene were digested by endonucleases EcoRⅠ and NotⅠ. The insert and vector fragments were extracted and ligated to construct the pEZ-LAG3-mCherry plasmid. The pEZ-LAG3-mCherry plasmid was sequenced and transfected into the HEK293T cells using Lipofectamine 3000. The expression location of LAG3-mCherry in the HEK293T cells was observed with fluorescence microscope and the expression of LAG3-mCherry fusion proteins in the cells were detected by Western blotting method.

The results of double digestion of recombinant plasmid showed that two DNA bands of about 8 012 and 2 066 bp were found in gel electrophoresis， which were consistent with the size of pEZ-Lv-mCherry vector and LAG3 gene fragment. The DNA sequencing results showed that the LAG-3 gene was successfully inserted into the lentiviral expression vector. The fluorescence microscope results demonstrated that most of LAG3-mCherry proteins were located on the cell membrane of HEK293T cells， while a few fusion proteins were found in the cytoplasm area. The results of Western blotting method showed a specific protein band of LAG3 in the HEK293T cells transfected with pEZ-LAG3-mCherry plasmid.

The pEZ-LAG3-mCherry plasmid including LAG3-mCherry DNA sequence is successfully constructed and the cell line stably expressing LAG3-mCherry is established. The LAG3-mCherry fusion protein is mainly distributed on the cell membrane of HEK293T cells transfected with pEZ-LAG3-mCherry plasmid.