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Construction of xenoantigen α-1,3GT gene expression vector regulated by hTERT promoter and its targeting expression in human lung cancer cells
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Abstract:
Objective:To construct an xenoantigen synthetase α1,3 galactosyltransferase (α1,3GT) eukaryotic expression vector regulated by human telomerase catalytic subunit (hTERT) promoter, and to investigate its targeting expression of α1,3GT in lung cancer cell lines. Methods: Previously prepared and confirmed pig α1,3GT gene was inserted into pEGFPhTERTp plasmid to construct eukaryotic expression vector pEGFPhTERTpGT. pEGFPhTERTpGT and pEGFPN1GT (α1,3GT eukaryotic expression vector under the control of CMV promoter) were transfected into telomerasepositive human lung adenocarcinoma A549 cells and telomerasenegative human embryonic lung fibroblast MRC5 cells. 1,3GT mRNA expression in the transfected cells was detected by RTPCR. Expression of αgal antigen in transfected cells was examined by immunofluorescence and flow cytometry. Results: pEGFPhTERTpGT eukaryotic expression vector was successfully constructed. Both A549 and MRC5 cells transfected with pEGFPN1GT showed expression of α1,3GTmRNA; A549 cells but not telomerasenegative MRC5 cells expressed α1,3GT mRNA after transfection with pEGFPhTERTpGT. Furthermore, both A549 and MRC5 cells transfected with pEGFPN1GT showed expression of xenoantigen αgal; A549 but not MRC5 cells expressed xenoantigen αgal after transfection with pEGFPhTERTpGT (P<0.01). Conclusion: α1,3GT gene under the regulation of hTERT promoter can be specifically expressed in telomerasepositive lung cancer cell lines, which can induce production of xenoantigen αgal.
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Project Supported:
Supported by the National Natural Science Foundation of China (No. 30270589, No. 30470762)
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