本文關(guān)鍵詞：GRP78基因過表達(dá)載體的構(gòu)建及在人臍靜脈內(nèi)皮細(xì)胞的表達(dá)　出處：《上海交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2016年06期 　論文類型：期刊論文

  更多相關(guān)文章： 葡萄糖調(diào)節(jié)蛋白 質(zhì)粒構(gòu)建 人臍靜脈內(nèi)皮細(xì)胞 內(nèi)質(zhì)網(wǎng)

【摘要】：目的構(gòu)建葡萄糖調(diào)節(jié)蛋白78(GRP78)基因過表達(dá)質(zhì)粒,體外轉(zhuǎn)染人臍靜脈內(nèi)皮細(xì)胞(HUVECs)并觀察其表達(dá)和定位。方法根據(jù)Pub Med數(shù)據(jù)庫中GRP78基因信息,設(shè)計(jì)引物,采用PCR擴(kuò)增其基因片段;運(yùn)用基因重組方法雙酶切目的基因,并分別將其克隆至含有綠色熒光蛋白(GFP)的p LVX-IRES-Zs Green1載體以及含有Flag標(biāo)簽的CMV-10載體,經(jīng)酶切、測(cè)序?qū)χ亟M質(zhì)粒進(jìn)行鑒定。轉(zhuǎn)染成功構(gòu)建的質(zhì)粒至HUVECs,免疫熒光染色法觀察蛋白表達(dá)和定位,Western blotting驗(yàn)證蛋白過表達(dá)情況。結(jié)果酶切和測(cè)序鑒定表明成功構(gòu)建p LVX-GRP78-IRES-Zs Green1以及CMV-Flag-GRP78的基因重組質(zhì)粒;將成功構(gòu)建的p LVX-GRP78質(zhì)粒轉(zhuǎn)染至HUVECs,可觀察到大量綠色熒光表達(dá);將成功構(gòu)建的Flag-GRP78質(zhì)粒轉(zhuǎn)染至HUVECs,免疫熒光檢測(cè)表明Flag-GRP78蛋白表達(dá)定位于內(nèi)質(zhì)網(wǎng);Western blotting證實(shí)p LVX-GRP78和Flag-GRP78成功過表達(dá)。結(jié)論成功構(gòu)建p LVX-GRP78、Flag-GRP78重組質(zhì)粒,轉(zhuǎn)染HUVECs并觀察到其內(nèi)質(zhì)網(wǎng)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.
【作者單位】： 蚌埠醫(yī)學(xué)院;上海交通大學(xué)醫(yī)學(xué)院附屬第九人民醫(yī)院內(nèi)分泌科;
【基金】：國家自然科學(xué)基金(81270908)~~
【分類號(hào)】：Q78;R3416
【正文快照】： 葡萄糖調(diào)節(jié)蛋白78(glucose-regulated protein 78,GRP78)又稱免疫球蛋白重鏈結(jié)合蛋白,是發(fā)現(xiàn)最早的一類內(nèi)質(zhì)網(wǎng)伴侶蛋白,屬于熱休克蛋白70(heatshock protein 70,Hsp70)家族的一員,與HSP70家族其他成員氨基酸序列有高度同源性,且在進(jìn)化中高度保守[1-2]。當(dāng)機(jī)體受到外傷、缺氧、

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1 鈕曉音;李小彥;陳廣潔;;綠色熒光蛋白標(biāo)記的GRP78蛋白表達(dá)和功能研究[J];南通醫(yī)學(xué)院學(xué)報(bào);2009年04期

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