Protein transduction domain-hA20 fusion protein protects endothelial cells against high glucose-induced injury
Protein transduction domain-hA20 fusion protein protects endothelial cells against high glucose-induced injury
复制标题
蛋白转导结构域-hA20融合蛋白保护内皮细胞免受高糖诱导的损伤
DOI：
10.4238/2012.july.19.9
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发表时间：
2012-01-01
影响因子：
0.4
通讯作者：
Zhou, Z. H.
中科院分区：
文献类型：
--
作者：
Hou, C. L.;Huang, Q.;Zhou, Z. H.
我们构建了一个含有蛋白转导结构域（PTD）和人A20（hA 20）基因片段的质粒，通过在毕赤酵母GS115中高效表达获得融合蛋白。通过添加9 xArg和EcoR获得质粒。在引物的末端添加识别位点，以及6xHis-标签和Not。识别站点到其结束。测序后将hA 20基因片段插入质粒pPIC 9 k中，构建表达载体pPIC 9 k-PTD-hA 20，转染GS115，诱导PTD-hA 20蛋白表达。我们使用镍柱从酵母发酵上清液中纯化蛋白质。将人脐静脉内皮细胞（HUVEC）在高葡萄糖培养基（30 mM葡萄糖）和含有不同浓度蛋白质的高葡萄糖培养基中培养。72 h后用TUNEL法检测HUVECs凋亡情况。生物学活性测试表明，该融合蛋白不仅能自由地穿过细胞膜，而且能抑制高糖诱导的HUVECs凋亡。结论：PTD-hA 20融合蛋白具有潜在的临床应用价值。
We constructed a plasmid containing a protein transduction domain (PTD) and a human A20 (hA20) gene fragment; the fusion protein was obtained by highly expressing this plasmid in the yeast Pichia pastoris GS115. The plasmid was obtained by adding 9xArg and EcoR. recognition sites to the end of the primer, and 6xHis-Tag and Not. recognition sites to its end. After sequencing, the hA20 gene fragment was inserted into plasmid pPIC9k to construct expression vector pPIC9k-PTD-hA20; then, we transfected GS115 with the vector and induced PTD-hA20 protein expression. We purified protein from the yeast fermentation supernatant using a nickel column. Human umbilical vein endothelial cells (HUVECs) were cultured in high glucose medium (30 mM glucose) and in high glucose medium containing different concentrations of protein. Apoptosis of HUVECs was assayed by TUNEL 72 h later. The biological activity tests indicated that the fusion protein not only passed through the cell membrane freely, but also inhibited apoptosis of HUVECs induced by high glucose levels. We conclude that the fusion protein PTD-hA20 has potential for clinical use.