Abstract：Objective: To construct the lentiviral and eukaryotic expression systems of mouse T cell Ig and ITIM domin(TIGIT) cell transmembrane protein fused with mLumin fluorescent protein as an indicator system and to obtain mTIGIT-mLumin fusion protein for the development of a monoclonal antibody product. Methods: The gene fragments encoding mLumin or mTIGIT which containing extracellular and transmembrane regions, were amplified by PCR with special primers from existed plasmid or mouse genomic DNA, and inserted into plenti-puro and pcDNA3.1 vectors respectively. The recombinant vector or lentivirus packaging in HEK-293T cells with plasmid pMD2.0G were transfected into African green monkey kidney fibroblast Cos7 cells and human renal epithelial 293T cell respectively. Stable cell lines were selected with G418(geneticin) and purimycin respectively; fluorescence microscope and confocal microscope were used to detect the mLumin expression. Results: mTIGIT and mLumin were successfully amplified by PCR, and digested, inserted into and ligated with the vector subsequently. The sequence of the cloned mTIGIT and mLumin fragments was proved to be correct and inserted into the plenti-puro and pcDNA3.1 vectors successfully. Fluorescence and confocal microscopy observation showed that transfected HEK-293T cells and Cos7 cells could stably express mTIGIT-mLumin fusion protein after screening with G418 or purimycin respectively. Conclusion: The expression vectors of mTIGIT-mLumin were constructed correctly and the fusion protein were expressed stably in cells.
［Key words］T cell Ig and ITIM domin; mLumin; recombinant plasmid; fusion protein
2008, Vol. 28 
