人真核表达质粒载体pcDNA3/HA-moesin的构建及其在内皮细胞中的表达  

Construction of eukaryotic expression plasmid pcDNA3/HA-moesin and its expression in endothelial cells

作　　者：刘红霞[1] 罗卓章[1] 杜静[2] 郭晓华[2] 黄巧冰[2] 

机构地区：[1]佛山市南海区第二人民医院内分泌科,广东佛山528251 [2]南方医科大学病理生理学教研室,广东省医学休克微循环重点实验室,广东广州510515

出　　处：《海南医学院学报》2014年第1期1-4,7,共5页Journal of Hainan Medical University

基　　金：国家自然科学基金(资助项目30771028);教育部创新团队项目(IRT0730)~~

摘　　要：目的:构建人膜突蛋白(moesin)的真核表达质粒pcDNA3/HA-moesin,并检测其在内皮细胞中的表达。方法:采用RT-PCR法从人脐静脉内皮细胞株(HUVECs)中克隆得到moesin cDNA全长序列,经双酶切后连接到真核表达载体pcDNA/HA中,挑选出的阳性克隆经双酶切和测序鉴定后,经脂质体转染法转入HUVECs中,采用Realtime PCR及Western blot等方法检测目的基因mRNA及蛋白的表达。结果:pcDNA3/HA-moesin质粒经双酶切鉴定和DNA测序证实,目的基因moesin的序列完全正确,真核表达质粒构建成功。Realtime PCR检测显示,转染pcDNA3/HA-moesin组的moesin mRNA表达水平明显高于未转染组和转染pcDNA3/HA空载体组,后2组间无明显差异。Western blot结果显示转染pcDNA3/HA-moesin组有HA-moesin蛋白表达,而未转染组和转染pcDNA3/HA空载体组未检测到HA-moesin表达。结论:成功构建pcDNA3/HA-moesin真核表达载体,在体外转染HUVECs后可使moesin mRNA表达增加,并成功表达HA-moesin,为进一步鉴定内皮细胞中晚期糖基化终产物(AGEs)引起的moesin的磷酸化位点奠定了实验基础。Objective：To construct the eukaryotic expression plasmid of moesin,and to detect the expression of the plasmid in endothelial cells.Methods：The full-length cDNA of moesin was cloned from HUVECs by RT-PCR and was ligated into eukaryotic expression vector pcDNA3/HA to obtain recombinant plasmid pcDNA3/HA-moesin,which was identified by EcoR/XhoI double digestion and nucleotide sequencing.Finally,the recombinant plasmid was transfected into HUVECs by LipofectamineT M LTX and PLUST Mreagents.The expression levels of moesin mRNA and HA-moesin protein were detected respectively by Realtime PCR and Western blot.Results：Recombinant plasmid pcDNA3/HA-moesin was proved to be successfully constructed by restriction enzyme digestion analysis with EcoR/XhoI and DNA sequencing,and the sequence of target gene was completely correct.Realtime PCR showed that the expression of moesin mRNA in pcDNA3/HA-moesin transfection group was higher than that in non-transfected and pcDNA3/HA transfection group,while no difference existed between the latter two groups.Western blot detected the protein expression of HA-moesin in transfected pcDNA3/HA-moesin group,but not detected in non-transfected and pcDNA3/HA transfection group.Conclusion：Eukaryotic expression plasmid pcDNA3/HA-moesin has been successfully constructed,and the expression levels of moesin mRNA is increased,pcDNA3/HA-moesin expressed HA-moesin protein in HUVECs,this will facilitate further functional study of moesin gene in HUVECs.

关 键 词：人膜突蛋白 真核表达质粒 人脐静脉内皮细胞株 

分 类 号：R282.71[医药卫生—中药学]