[1]左玉梅,王媛,刘建平.真核表达载体pEGFP-N2/AT2R的构建及其在原代血管平滑肌细胞的表达[J].第三军医大学学报,2011,33(09):940-943.
 Zuo Yumei,Wang Yuan,Liu Jianping.Construction of eukaryotic expressing vector pEGFP-N2/AT2R and its expression in primary vascular smooth muscle cells[J].J Third Mil Med Univ,2011,33(09):940-943.
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真核表达载体pEGFP-N2/AT2R的构建及其在原代血管平滑肌细胞的表达(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
33卷
期数:
2011年第09期
页码:
940-943
栏目:
论著
出版日期:
2011-05-15

文章信息/Info

Title:
Construction of eukaryotic expressing vector pEGFP-N2/AT2R and its expression in primary vascular smooth muscle cells
作者:
左玉梅王媛刘建平
第三军医大学:西南医院心血管内科,重庆市介入心脏病学研究所,训练部教务处
Author(s):
Zuo YumeiWang YuanLiu Jianping
Department of Cardiology, Southwest Hospital,Office of Division of Educational Affairs, Third Military Medical University, Chongqing, 400038, China
关键词:
血管紧张素Ⅱ2型受体基因克隆原代血管平滑肌细胞绿色荧光蛋白
Keywords:
Ang Ⅱ type 2 receptor cloning primary vascular smooth muscle cells enhanced green fluorescent protein
分类号:
R394-33; R322.12; R394.2
文献标志码:
A
摘要:
目的    体外构建携带血管紧张素Ⅱ2型受体(ANG Ⅱ Type 2  Receptor,AT2R)基因的增强型绿色荧光蛋白真核表达载体,并观察其在大鼠血管平滑肌细胞(vascular smooth muscle cells, VSMCs)中的表达。     方法      以pUHD-10.3/AT2R质粒为模板,PCR扩增AT2R基因的全长cDNA序列, 再将其克隆入载体pEGFP-N2,构建其真核表达载体pEGFP-N2/AT2R。以基因转染技术,将AT2R导入原代VSMCs。倒置荧光显微镜观测转染后VSMCs生长变化及AT2R在其中的表达等情况。图像分析技术检测AT2R在VSMCs中的转染效率。Western blot检测转染AT2R基因的VSMCs表达其编码蛋白。RT-PCR法对AT2R基因修饰的VSMCs进行鉴定。    结果     成功构建AT2R基因的真核表达载体pEGFP-N2/AT2R,该真核载体能携带AT2R基因转染并有效表达于VSMCs,其转染效率约40%,RT-PCR及Western blot均可检测到AT2R mRNA及蛋白在血管平滑肌细胞中高表达。    结论      成功地将克隆到的AT2R基因克隆入pEGFP-N2载体中,并实现了AT2R基因在原代VSMCs的表达。
Abstract:
Objective    To clone Ang Ⅱ type 2 receptor (AT2R) gene, obtain enhanced green fluorescent protein(EGFP) vector for the gene, and then observe its expression in primary vascular smooth muscle cells (VSMCs) after transfection.     Methods     Utilizing plasmid pUHD-10.3/AT2R as a template, the full length cDNA fragment of AT2R was amplified by polymerase chain reaction (PCR). Eukaryotic expression vector pEGFP-N2/AT2R was constructed by cloning AT2R gene into vector pEGFP-N2. After transfecting AT2R into primarily cultured VSMCs, invert fluorescent microscopy was employed to observe VSMCs growth as well as AT2R expression in VSMCs. Transfection efficiency of AT2R in VSMCs was evaluated by image analyzor, the protein and mRNA expressions of AT2R in VSMCs were examined by Western blotting and RT-PCR.     Results     Eukaryotic expression vector pEGFP-N2/AT2R was successfully constructed. The eukaryotic vector expressing AT2R gene was efficiently transfected and expressed in VSMCs. The transfection efficiency of AT2R gene in VSMCs was about 40%. RT-PCR and Western blotting indicated that AT2R was expressed in cultured VSMCs at mRNA and protein levels.     Conclusion       cDNA sequence encoding AT2R gene is successfully cloned into pEGFP-N2 which can be efficiently expressed in primary VSMCs.

参考文献/References:

左玉梅, 王媛, 刘建平. 真核表达载体pEGFP-N2/AT2R的构建及其在原代血管平滑肌细胞的表达[J].第三军医大学学报,2011,33(9):940-943.

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更新日期/Last Update: 2011-05-04