摘要
目的:利用基因工程技术构建人Wnt10b基因的pEGFP-N1-Wnt10b真核表达载体,观察其在COS-7细胞中的表达。方法:以pUC19-Wnt10b为模板,PCR扩增人Wnt10b基因的cDNA编码区序列,扩增产物和pEGFP-N1载体双酶切,T4 DNA连接酶连接,构建pEGFP-N1-Wnt10b,酶切和测序鉴定该载体;LipofectamineTM2000将该载体转染入COS-7中,Western blot和免疫细胞化学检测COS-7 Wnt10b蛋白表达。结果:PCR扩增出的人Wnt10b基因cDNA正确克隆到pEGFP-N1载体,成功构建了pEGFP-N1-Wnt10b;将其转染入COS-7,COS-7 Wnt10b蛋白表达明显增高。结论:成功构建了人pEGFP-N1-Wnt10b,这为进一步研究Wnt10b基因功能奠定了前期基础。
Objective：To construct human Wnt10b gene eukaryotic expression vector,and to observe its expression in transient transfeted COS-7 cell line.Methods：cDNA fragment was amplified from pUC19-Wnt10b by PCR and cloned into eukaryotic expression vector pEGFP-N1.The positive clone was confirmed by enzyme digestion and sequencing.The recombinant plasmid was transiently transfected into COS-7 cell line with LipofectamineTM2000.The expression of Wnt10b gene was detected by Western blot and immunocytochemistry.Results：Eukaryotic expression vectors containing coding region of human Wnt10b gene was constructed.COS-7 cells transfected with the recombinant plasmid expressed high level of Wnt10b protein in cytoplasm.Conclusion：pEGFP-N1-Wnt10b is constructed,which can provide a strong molecular tool for further studies on Wnt10b gene function.
出处
《汕头大学医学院学报》
2011年第4期196-199,F0002,共5页
Journal of Shantou University Medical College
基金
国家自然科学基金资助项目(81071577)
广东省自然科学基金资助项目(9151008901000195)
广东省科技计划资助项目(2009B030801320)
广东省医学科研基金资助项目(A2009433)
