GRP78基因过表达载体的构建及在人脐静脉内皮细胞的表达

发布时间：2017-12-28 19:01

本文关键词：GRP78基因过表达载体的构建及在人脐静脉内皮细胞的表达　出处：《上海交通大学学报(医学版)》2016年06期 　论文类型：期刊论文

【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Pub Med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p LVX-IRES-Zs Green1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p LVX-GRP78-IRES-Zs Green1以及CMV-Flag-GRP78的基因重组质粒;将成功构建的p LVX-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p LVX-GRP78和Flag-GRP78成功过表达。结论成功构建p LVX-GRP78、Flag-GRP78重组质粒,转染HUVECs并观察到其内质网定位。
[Abstract]:Objective to construct the overexpression plasmid of glucose regulated protein 78 (GRP78) gene, and to transfect human umbilical vein endothelial cells (HUVECs) in vitro and observe its expression and localization. Methods according to the GRP78 Pub Med database of genetic information, design primers, amplified by PCR gene fragment using gene recombination method; double enzyme gene, and cloned into a green fluorescent protein (GFP) were p LVX-IRES-Zs Green1 CMV-10 vector and Flag vector containing the label by enzyme digestion and sequencing to identify recombinant plasmid. The transfected plasmids were successfully constructed to HUVECs, and the protein expression and localization were observed by immunofluorescence staining, and the overexpression of the protein was verified by Western blotting. The results of enzyme digestion and DNA sequencing revealed the successful construction of P LVX-GRP78-IRES-Zs and Green1 CMV-Flag-GRP78 gene recombinant plasmid; P successfully constructed LVX-GRP78 plasmid was transfected into HUVECs, was observed in large green fluorescence; successfully constructed Flag-GRP78 plasmid was transfected into HUVECs, immunofluorescence assay showed that Flag-GRP78 protein located in endoplasmic reticulum; Western blotting confirmed P LVX-GRP78 and Flag-GRP78 successfully expressed. Conclusion the recombinant plasmid of P LVX-GRP78 and Flag-GRP78 was successfully constructed, HUVECs was transfected and the localization of endoplasmic reticulum was observed.
【作者单位】：蚌埠医学院;上海交通大学医学院附属第九人民医院内分泌科;
【基金】：国家自然科学基金(81270908)~~
【分类号】：Q78;R3416
【正文快照】： 葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)又称免疫球蛋白重链结合蛋白,是发现最早的一类内质网伴侣蛋白,属于热休克蛋白70(heatshock protein 70,Hsp70)家族的一员,与HSP70家族其他成员氨基酸序列有高度同源性,且在进化中高度保守[1-2]。当机体受到外伤、缺氧、

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