过表达三突变型低氧诱导因子1α重组人脐静脉内皮细胞株的构建及鉴定  

Construction and identification of recombinant human umbilical vein endothelial cell line overexpressing triple mutant hypoxia-inducible factor-1α

作　　者：李慧[1] 杨鹏程[2] 韦莉莉[1] 李桥川[2] LI Hui;YANG Peng-cheng;WEI Li-li;LI Qiao-chuan(Department of Geriatric Cardiology;Department of Hematology,the First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学第一附属医院老年综合科,南宁市530021 [2]广西医科大学第一附属医院血液内科,南宁市530021

出　　处：《广西医学》2018年第11期1224-1227,共4页Guangxi Medical Journal

基　　金：国家自然科学基金(81360036)

摘　　要：目的构建并鉴定过表达三突变型低氧诱导因子1α(HIF-1α)重组人脐静脉内皮细胞株EA.hy926。方法根据原有质粒pc DNA3.1+-HIF1-Ala402-Ala564-Ala803的HIF-1α三突变基因序列设计引物,经PCR扩增后连接至入门载体,将含有目的基因的入门载体与p T590质粒及p ENTR_L4_PCMV_R1质粒拼接为三突变型HIF-1α重组慢病毒载体,将该载体转化至DH5α感受态细胞,挑选阳性的克隆测序鉴定。将三突变型HIF-1α重组慢病毒载体与辅助包装质粒共转染人胚肾细胞系HEK293T细胞,收集病毒液。将EA.hy926细胞分为感染重组HIF-1α过表达慢病毒的Lenti-HIF-1α组、感染阴性对照慢病毒的Lenti-negative组、只加培养基的MOCK组,给予相应干预后采用嘌呤霉素(1μg/ml)筛选高表达HIF-1α细胞株,观察病毒感染效率,采用蛋白免疫印迹法分析HIF-1α蛋白表达情况。结果测序鉴定结果提示成功合成三突变型HIF-1α重组质粒。荧光显微镜下观察到HEK293T细胞中有大量增强绿色荧光蛋白(EGFP)表达。重组慢病毒感染EA.hy926细胞后,荧光显微镜下可见大量EGFP表达,Lenti-HIF-1α组细胞稳定表达HIF-1α蛋白,且其表达水平高于Lenti-negative组及MOCK组。结论重组三突变型HIF-1α慢病毒过表达载体可成功构建,并可获得稳定表达HIF-1α的人脐静脉内皮细胞株。Objective To construct and identify recombinant human umbilical vein endothelial cell line EA.hy926 overexpressing triple mutant hypoxia-inducible factor 1α（HIF-1α）. Methods Primers design was conducted according to triple mutant HIF-1α sequence of plasmid pcDNA3.1 ＋-HIF1-Ala402-Ala564-Ala803,and the gene segment was linked to the entry vector after amplification by PCR. The recombinant lentiviral vector with triple mutant HIF-1α was constructed by combining the entry vector carrying target gene,plasmid pT590 and pENTRL4PCMVR1,then was transformed into DH5α competent cells.The positive clones were collected and sequenced for identification. The recombinant lentiviral vector with triple mutant HIF-1α and the auxiliary packaging plasmid were cotransfected into HEK293T cells,then the fluid containing virus was collected.The EA.hy926 cells were divided into Lenti-HIF-1α group infected by recombinant lentivirus overexpressing HIF-1α,Lenti-negative group infected by negative control lentivirus,and MOCK group added with medium.The cell lines overexpressing HIF-1α were screened with 1 μg/ml puromycin after corresponding intervention,then the efficiency of viral infection was observed,and the expression of HIF-1α protein was analyzed by Western Blot assay. Results The result of sequencing and identification indicated that recombinant plasmid with triple mutant HIF-1α was successfully synthesized.High-expression of enhanced green fluorescent protein（EGFP） was observed in the HEK293T cells under fluorescence microscope.After infection by recombinant lentivirus,high-expression of EGFP was observed in the EA.hy926 cells under fluorescence microscope.The HIF-1α protein was stably expressed in the cells of Lenti-HIF-1α group and was higher than that in the cells of the Lenti-negative group or the MOCK group. Conclusion The recombinant lentiviral vector overexpressing triple mutant HIF-1α was successfully constructed,and the human umbilical vein endothelial cell line stably overexpressing HIF-1α was

关 键 词：低氧诱导因子1Α 三突变 过表达 慢病毒载体 人脐静脉内皮细胞 

分 类 号：R34[医药卫生—基础医学]