Construction of xenoantigen α-1,3GT gene expression vector regulated by hTERT promoter and its targeting expression in human lung cancer cells

Objective:To construct an xenoantigen synthetase α-1,3 galactosyltransferase(α-1,3GT) eukaryotic expression vector regulated by human telomerase catalytic subunit(hTERT) promoter,and to investigate its targeting expression of α-1,3GT in lung cancer cell lines.Methods:Previously prepared and confirmed pig α-1,3GT gene was inserted into pEGFP-hTERTp plasmid to construct eukaryotic expression vector pEGFP-hTERTp-GT.pEGFP-hTERTp-GT and pEGFP-N1-GT(α-1,3GT eukaryotic expression vector under the control of CMV promoter) were transfected into telomerase-positive human lung adenocarcinoma A549 cells and telomerase-negative human embryonic lung fibroblast MRC-5 cells.1,3-GT mRNA expression in the transfected cells was detected by RT-PCR.Expression of α-gal antigen in transfected cells was examined by immunofluorescence and flow cytometry.Results:pEGFP-hTERTp-GT eukaryotic expression vector was successfully constructed.Both A549 and MRC-5 cells transfected with pEGFP-N1-GT showed expression of α-1,3GT mRNA;A549 cells but not telomerase-negative MRC-5 cells expressed α-1,3GT mRNA after transfection with pEGFP-hTERTp-GT.Furthermore,both A549 and MRC-5 cells transfected with pEGFP-N1-GT showed expression of xenoantigen α-gal;A549 but not MRC-5 cells expressed xenoantigen α-gal after transfection with pEGFP-hTERTp-GT(P0.01).Conclusion:α-1,3GT gene under the regulation of hTERT promoter can be specifically expressed in telomerase-positive lung cancer cell lines,which can induce production of xenoantigen α-gal.