Abstract:

Objective To construct the lentiviral expression vector of T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain-green fluorescent protein （TIGIT-GFP） and establish a cell line stably expressing TIGIT-GFP， and to explore the expression of TIGIT-GFP fusion protein in stable expression cell line. Methods The TIGIT plasmid and lentiviral vector plenti-GFP were doubly digested by endonucleases EcoR Ⅰ and Not Ⅰ. The inserted gene and vector in agarose gel were recovered and ligated to construct the recombinant plasmid pLenti-TIGIT-GFP. The human embryonic kidney HEK293T cells transfected with sequenced recombinant plasmid were used as experimental group， and the HEK293T cells transfected with pCMV6-TIGIT were used as control group. After 48 h of transfection， the expression of GFP in the cell membrane was detected under fluorescence microscope. The cells in experimental group were cultured and screened with puromycin for two weeks； the screened cells were selected and expanded，and the expression of TIGIT-GFP protein in cell membrance was examined by fluorescence microscope. Western blotting method was used to determine the TIGIT-GFP protein expression in monoclonal cells. Results The enzyme digestion results showed that TIGIT gene was inserted into the expression vector pLenti-GFP，and the DNA sequencing showed no mutation.The GFP in the cell membrane in experimental group was found. The Western blotting results showed a specific band of TIGIT-GFP in cell lysis buffer in experimental group. Conclusion The lentiviral expression vector pLenti-TIGIT-GFP is successfully established， the cell line stably expression TIGIT-GFP fusion protein is constructed，and the TIGIT-GFP protein is mainly expressed in the cell membrane of the stable cell line.

Key words: T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain, Green fluorescent protein, Lentivirus expression vector, Stable transfection, Monoclonal cells