Abstract: Objective: To construct the eukaryotic expression vector of signal regulatory protein α-green fluorescent protein (SIRPα-GFP) to transfer the HEK293T cells to establish a cell line stably expressing SIRPα-GFP fusion protein,and to investigate the membrane location of SIRPα-GFP fusion protein in the HEK293T cells. Methods: The SIRPα plasmid and the expression vector including GFP were double-digested by endonucleases MluⅠand SgfⅠ, respectively.The SIRPα gene was cloned into the pLenti-GFP vector to construct the pLenti-SIRPα-GFP plasmid.The pLenti-SIRPα-GFP plasmid was transformed into the DH5α E. coli competent cells and sequenced. The plasmids were transfected into the HEK293T cells by Lipofectamine 3000.The expression of SIRPα-GFP in the stably transfected HEK293J cells was observed by fluorescence microscope and the expression of SIRPα-GFP fusion protein in HEK293T cells was detected by Western blotting method. Results: The results of enzyme digestion and DNA sequencing showed that the recombinant plasmid pLenti-SIRPα-GFP was successfully constructed. The fluorescence microscope analysis showed that the SIRPα-GFP fusion protein was located on the membrane of HEK293T cells. The Western blotting results confirmed the successful expression of SIRPα protein in the HEK293T cells transfected with pLenti-SIRPα-GFP plasmid. Conclusion: The pLenti-SIRPα-GFP plasmid is successfully constructed. The cell line stably expressing SIRPα-GFP is successfully prepared by transfecting the pLenti-SIRPα-GFP plasmid into the HEK293T cells. The SIRPα-GFP protein is located on the membrane of HEK293T cells.

Key words: signal regulatory protein α, green fluorescent protein, eukaryotic expression vector, cell transfection