人HoxA10基因真核表达载体构建和表达产物的鉴定

目的:克隆人子宫内膜容受性标志分子HoxA10基因的cDNA,转入真核细胞载体,并在293T细胞中进行表达和鉴定。方法:应用RT-PCR法从人子宫内膜组织中扩增HoxA10基因的cDNA编码区序列,将PCR产物克隆至pCMV-HA真核表达载体中,测序鉴定该载体。以LipofectamineTM2000将该载体转染入293T细胞中,Western blotting检测HoxA10蛋白的表达。结果:PCR扩增出的人HoxA10基因cDNA正确克隆到了pCMV-HA载体,成功构建了pCMV-HA-HoxA10重组载体;将其转染入293T细胞,用HoxA10和HA抗体进行Western blotting,均检测到了融合蛋白的表达。结论:携带有HA蛋白标签的人HoxA10基因的真核表达载体pCMV-HA-HoxA10克隆及表达成功,为进一步研究HoxA10与其它蛋白质的相互作用以及建立HoxA10的荧光素酶报告基因分析系统奠定了基础。

Construction and Identification of Eukaryotic Expression Vector Bearing Human HoxA10 Gene

Objective: To clone the cDNA of human HoxA10 gene,and to construct eukaryotic expression vector of HoxA10 for expression and identification in 293T cell line.Methods: cDNA fragment of HoxA10 was amplified from human endometrium by RT-PCR and cloned into eukaryotic expression vector pCMV-HA.The positive clone was confirmed by sequencing.The recombinant plasmid was transiently transfected into 293T cell line with Lipofectamine TM 2000.The protein expression of HoxA10 was detected by Western blotting.Results: Eukaryotic expression vector pCMV-HA-HoxA10 containing coding region of human HoxA10 gene was successfully constructed.293T cells transfected with the recombinant plasmid expressed high level of recombinant HoxA10 protein detected by Western blotting using HoxA10 and HA antibodies.Conclusion: The construction and the expression of pCMV-HAHoxA10 were achieved successfully,which has prepared for the experimental ground for further studies of HoxA10 interacting proteins and establishment of HoxA10 luciferase reporter assay system.