Cloning of human Gal-9 gene and its expression in CHO cells

Objective:To construct the eukaryotic expression vector of Gal-9 and to express it in CHO cells and detect its product.Methods:Gal-9 gene was obtained by PCR and DNA recombination from PBMC.PCR product of Gal-9 was then inserted into plasmid pcDNA3.1(+).The recombinant vector was identified by PCR,restriction enzyme digestion and sequencing.Using LipofectamineTM 2000,the plasmid pGal-9 was transfected into CHO and then detected by Western blot and RT-PCR.MTT colorimetry was used to detect allogeneic lymphocyte proliferation.Results:DNA sequencing and restriction enzyme digestion verified the correction of recombinant plasmid pGal-9.After plasmid pGal-9 was transfected into CHO,the expressed product of Gal-9 was detected by Western blot and RT-PCR.The result showed that Gal-9 significantly inhibited lymphocyte proliferation response,the level was 85.43%.Conclusion:The eukaryotic expression vector pGal-9 has been successfully constructed and successfully expressed in CHO cell line,and Gal-9 inhibited lymphocyte proliferation response.