Abstract:

To construct the eukaryotic expression vectors of human HMGB1 gene and investigate its immunomodulatory effect. MethodsThe HMGB1 gene fragment  was isolated from cloning vector pMDHMGB1 with digestion of Sal I and EcoR I and agarose gel separation, and inserted into the same sites of eukaryotic expression vector pCITEEGFP. The recombinant plasmid was screened by colony PCR and restriction enzyme digestion. The human monocyte cell line U937 was transfected with recombinant expression plasmid and screened by gradient G4l8. Expression of HMGB1 was determined under an inverted fluorescence microscope and Western blotting. ResultsColony PCR and the restriction endonuclease digestion showed that the HMGB1 gene was correctly inserted into the eukaryotic expression vector and the recombinant plasmid pCITEEGFPHMGB1 was obtained. With an increase of G418 concentration in the culture medium, the U937 cells stably expressing human HMGB1 were established. The quantity of HMGB1 in the transfected cells was increased. ConclusionThe eukaryotic vector of pCITEEGFPHMGB1 was successfully constructed and a human monocyte cell line stably expressing HMGB1 was obtained, which established a solid foundation for further study on the effects and mechanisms of HMGB1on mononuclearmacrophages.

Key words: High mobility group proteins； Eukaryotic cells； Monocytes