可溶性血管内皮细胞生长因子受体1真核表达载体pEGFP-N1/sflt-1构建及在人脐静脉内皮细胞中的表达  

Construction of soluble vascular endothelial growth factor receptor-1 eukaryotic expression vector pEGFP-N1/sflt-1 and its expression in human umbilical vein endothelial cells

作　　者：周仪华[1] 王耀晟[1] 何力鹏[1] 鲍晓明[1] 黄晓[1] 程晓曙[1] 

机构地区：[1]南昌大学第二附属医院心内科,江西省南昌市330006

出　　处：《中国组织工程研究与临床康复》2009年第41期8040-8043,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:研究表明,可溶性血管内皮生长因子受体Flt-1(sflt-1)基因在多种组织中表达,但其在内皮细胞中的表达尚待证实。目的:获取人sflt-1基因,构建sflt-1基因真核表达载体pEGFP-N1/sflt-1重组质粒,检测其在人脐静脉内皮细胞中的表达。设计、时间及地点:细胞学体外观察,于2008-05/12在南昌大学第二附属医院分子中心完成。材料:pEGFP-N1真核表达载体为南昌大学第二附属医院分子中心保存,人脐静脉内皮细胞株购至南京基凯生物技术公司。方法:从含有目的基因的质粒克隆模板中,利用酶切的方法获得目的基因,将目的载体进行酶切,纯化酶切产物后进行定向连接,其产物转化细菌感受态细胞,PCR鉴定为阳性的克隆,证明目的基因已经定向连入目的载体。再对PCR鉴定阳性的克隆进行测序和分析比对,比对正确的即为构建成功的融合蛋白表达质粒载体。用脂质体法将pEGFP-N1/sflt-1重组质粒转染人脐静脉内皮细胞。主要观察指标:①重组质粒的酶切鉴定结果。②重组质粒的测序鉴定结果。③荧光显微镜及蛋白免疫印迹检测sflt-1基因在人脐静脉内皮细胞中的表达。结果:①对PCR鉴定阳性的克隆进行测序和分析比对,与sflt-1基因片段全长2063bp理论值相符,证实pEGFP-N1/sflt-1重组质粒构建成功。②重组质粒送至上海捷瑞生物技术公司进行通测,测序结果证实插入片段序列与理论值完全一致。③荧光显微镜下可见重组质粒转染人脐静脉内皮细胞发绿色荧光,蛋白免疫印迹检测表明sflt-1基因能在人脐静脉内皮细胞中表达。结论:实验成功构建了携带人血管内皮生长因子受体Flt-1的重组pEGFP-N1/sflt-1真核表达质粒,并证实其在人脐静脉内皮细胞中的表达。BACKGROUND： Research shows that soluble vascular endothelial growth factor receptor Fit-1 （sflt-1） gene can express in many tissues, but its expression in human umbilical vein endothelial cells （hUVECs） is still not confirmed. OBJECTIVE： To obtain sflt-1 gene, construct eukaryotic expression vector pEGFP-N1/sflt-1 and observe its expression in human umbilical vein endothelial cells. DESIGN, TIME AND SETTING： The cytology observation in vitro was conducted at the Molecule Center of Second Affiliated Hospital of Nanchang University from May to December 2008. MATERIALS： The eukaryotic vector pEGFP-N1 was preserved at the Molecule Center of Second Affiliated Hospital of Nanchang University. HUVECs were purchased from Nanjing Keygen Biotechnology Company. METHODS： sflt-1 gene was obtained with enzyme digestion from clone vector containing target gene. Following digesting target vector and purifying target product, the product was directionally linked with each other and then transformed into competent cells. The positive clone identified by PCR proved that the target gene sfit-1 was linked into pEGFP-N1 vector. Positive clones identified by PCR were sequenced and analyzed, and the correct sequence contrasted was the fusion protein expression plasmid vector. Recombinant plasmid pEGFP-N1/sflt-1 was transfected into hUVECs with liposome method. MAIN OUTCOME MEASURES： ① The identification of recombinant plasmids after enzyme digestion. ② The result of recombinant plasmid sequence. ③The expression of sflt-1 in hUVECS identified by a fluorescence microscope and Western-blot method. RESULTS： ①After contrasting the sequence and analysis results of the positive clones identified by PCR, the sflt-1 DNA segment was about 2 063 bp, in accordance with the expectation result. It indicated that the recombinant plasmid pEGFP-N1/sflt-1 was constructed successfully. ②The recombinant plasmid was measured by Shanghai Jerry Biotechnology Company, and the result showed that the sequence of segmen

关 键 词：可溶性血管内皮生长因子受体1 克隆 构建 转染 

分 类 号：R392.114[医药卫生—免疫学]