目的：构建人端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）启动子调控的异种移植抗原α1,3半乳糖基转移酶(α1,3 galactosyltransferase, α1,3GT）基因真核表达载体，研究其调控的α1,3GT在肿瘤细胞中的靶向表达。方法：将前期克隆并测序正确的猪α1,3GT基因定向克隆到pEGFPhTERTp质粒中, 构建α1,3GT真核表达载体pEGFPhTERTpGT。分别将pEGFPhTERTpGT和CMV启动子调控的α1,3GT真核表达质粒pEGFPN1GT转染端粒酶阳性的人肺癌细胞A549及端粒酶阴性的正常人胚肺成纤维细胞MRC5。RTPCR检测转染细胞中α1,3GT mRNA的表达，免疫荧光法和流式细胞仪检测αgal抗原的表达。结果：成功构建了pEGFPhTERTpGT真核表达载体。转染pEGFPN1GT的A549和MRC5中均有α1,3GT mRNA的表达；转染pEGFPhTERTpGT的A549中有α1,3GT mRNA的表达，而端粒酶阴性的MRC5细胞中无α1,3GT mRNA的表达。转染pEGFPN1GT的A549和MRC5中均可表达异种移植抗原αgal；转染pEGFPhTERTpGT质粒的A549中有αgal的表达，而MRC5细胞中无αgal的表达（P<0.01）。结论：hTERT启动子调控的α1,3GT 基因能靶向性表达在端粒酶阳性的肺癌细胞中，并合成异种移植抗原αgal。

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 (P<0.01). 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.

国家自然科学基金资助项目（No. 30270589，No. 30470762）