上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

GRP78基因过表达载体的构建及在人脐静脉内皮细胞的表达

李园园1，2，张光亚2，陈凤玲2   

  1. 1.蚌埠医学院, 蚌埠 233000； 2.上海交通大学 医学院附属第九人民医院内分泌科， 上海 201999
  • 出版日期:2016-06-28 发布日期:2016-07-25
  • 通讯作者: 陈凤玲, 电子信箱: cfl1993@126.com。
  • 作者简介:李园园（1990—）， 女， 硕士生; 电子信箱: 15921125173@163.com。
  • 基金资助:

    国家自然科学基金（81270908）

Construction of the GRP78 over-expressed vector and its expression in human umbilical vein endothelial cells

LI Yuan-yuan1,2, ZHANG Guang-ya2, CHEN Feng-ling2   

  1. 1.Bengbu Medical College, Bengbu 233000, China； 2.Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201999, China
  • Online:2016-06-28 Published:2016-07-25
  • Supported by:

    National Natural Science Foundation of China, 81270908

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摘要：

目的 构建葡萄糖调节蛋白78(GRP78)基因过表达质粒，体外转染人脐静脉内皮细胞(HUVECs)并观察其表达和定位。方法 根据PubMed数据库中GRP78基因信息，设计引物，采用PCR扩增其基因片段；运用基因重组方法双酶切目的基因，并分别将其克隆至含有绿色荧光蛋白(GFP)的pLVX-IRES-ZsGreen1载体以及含有Flag标签的CMV-10载体，经酶切、测序对重组质粒进行鉴定。转染成功构建的质粒至HUVECs，免疫荧光染色法观察蛋白表达和定位，Western blotting验证蛋白过表达情况。结果 酶切和测序鉴定表明成功构建pLVX-GRP78-IRES-ZsGreen1以及CMV-Flag-GRP78的基因重组质粒；将成功构建的pLVX-GRP78质粒转染至HUVECs，可观察到大量绿色荧光表达；将成功构建的Flag-GRP78质粒转染至HUVECs，免疫荧光检测表明Flag-GRP78蛋白表达定位于内质网；Western blotting证实pLVX-GRP78和Flag-GRP78成功过表达。结论 成功构建pLVX-GRP78、Flag-GRP78重组质粒，转染HUVECs并观察到其内质网定位。

关键词: 葡萄糖调节蛋白78, 质粒构建, 人脐静脉内皮细胞, 内质网

Abstract:

Objective To construct the glucose-regulated protein 78 (GRP78) over-expressed plasmid, transfect human umbilical vein endothelial cells (HUVECs) in vitro, and observe its expression and location. Methods Primers were designed according to the gene information on GRP78 in PubMed, and the GRP78 gene was amplified by Polymerase Chain Reaction (PCR). By using the gene recombination technology, the double enzyme digested GRP78 gene was cloned into the pLVX-IRES-ZsGreen1 vector with GFP and the CMV-10 vector with Flag tag. Recombined plasmids were identified with enzyme digestion and sequencing and were transfected into HUVECs. Immunofluorescence assay was used to detect its expression and location. Western blotting was used to confirm its over-expression. Results The results of enzyme digestion and sequencing revealed that the recombinant plasmids pLVX-GRP78-IRES-ZsGreen1 and CMV-Flag-GRP78 were successfully constructed. After transfecting HUVECs with the successfully constructed plasmid pLVX-GRP78, a large amount of green fluorescence was observed. After transfecting HUVECs with plasmid Flag-GRP78, immunofluorescence assay revealed that Flag-GRP78 protein located in endoplasmic reticulum. Western blotting showed the successful over-expression of pLVX-GRP78 and Flag-GRP78. Conclusion The recombinant plasmids pLVX-GRP78-IRES-Green1 and CMV-Flag-GRP78 were successfully constructed and were transfected into HUVECs. Flag-GRP78 protein was observed to locate in endoplasmic reticulum.

Key words: glucose-regulated protein 78, plasmid construction, human umbilical vein endothelial cell,
endoplasmic reticulum