ZNF580与EGFP融合蛋白表达载体的构建及其在HEK293细胞的表达和定位

[目的]构建人ZNF580与增强型绿色荧光蛋白（EGFP）融合蛋白真核表达载体,转染细胞进行瞬时表达,分析ZNF580-EGFP融合蛋白在人胚肾HEK293细胞中的表达和亚细胞定位。[方法]利用PCR技术扩增ZNF580基因cDNA开放阅读框架全编码区（编码1-172位氨基酸）、N端编码区（编码1-93位氨基酸）、C端编码区（编码94-172位氨基酸）,分别克隆到真核表达载体pEGFP-C1,BglΠ及HindШ双酶切筛选鉴定并测序。荧光显微镜下观察ZNF580-EGFP在HEK293细胞中的表达及亚细胞定位。[结果]pEGFP-ZNF580（1-172）、pEGFP-ZNF580（94-172）表达的ZNF580-EGFP融合蛋白聚集于HEK293细胞核。[结论]ZNF580蛋白的核定位信号位于其C端C2H2型锌指主构域（94-172位氨基酸区间）。

绿色荧光蛋白报告基因与ZNF580基因重组真核表达载体的构建与鉴定

【目的】构建具有绿色荧光蛋白报告 (GFP)基因的ZNF5 80真核表达载体 ,为转染细胞提供基础。【方法】根据ZNF5 80cDNA序列的开读框架设计上、下游引物 ,利用PCR技术扩增ZNF5 80cDNA开放阅读框序列 ,将PCR目的片段与PGEM T载体连接并克隆 ,酶切含目的片段PGEM T质粒及 pEGFP C1载体 ,回收纯化后做定向克隆连接 ,BglⅡ及HindⅢ双酶切电泳筛选、鉴定并测序。【结果】酶切 pEGFP C1 ZNF5 80 ,电泳分析插入片段长度为 :5 2 4bp ,与预期的结果一致 ,经连接点两端进行测序 ,测序结果显示接头两端序列连接正确。【结论】成功构建真核表达载体pEGFP C1 ZNF5 80。

ZNF580基因对人脐静脉内皮细胞增殖和迁移的影响

【目的】研究人C2H2型锌指蛋白基因ZNF580在人脐静脉内皮细胞增殖和迁移中的作用。【方法】利用细胞转染技术获得瞬时过表达ZNF580的EA.hy926内皮细胞,并用半定量RT-PCR、Western blotting法检测ZNF580的表达情况;采用MTT比色法和细胞迁移实验检测ZNF580对内皮细胞功能的影响。【结果】获得瞬时过表达ZNF580的内皮细胞(转染效率为60%),且ZNF580基因过表达内皮细胞增殖和迁移能力分别增强3倍和2.5倍。【结论】ZNF580能够促进内皮细胞增殖和迁移。

ZNF580基因原核表达载体的构建与鉴定

【目的】构建ZNF580基因原核表达载体,为获得大量重组ZNF580蛋白并制备其多克隆抗体奠定基础。【方法】根据ZNF580cDNA序列的开读框架设计上、下游引物,利用PCR技术扩增ZNF580开放阅读框cD-NA序列,将PCR扩增的目的片段与载体pET30a分别双酶切、回收纯化后做定向连接,EcoRI、XhoI双酶切电泳筛选、鉴定并测序。【结果】双酶切pET30a-ZNF580,电泳分析插入片段长度为524 bp,与预期的结果一致,经连接点两端进行测序,测序结果显示接头两端序列连接正确。【结论】成功构建原核表达载体pET30a-ZNF580。

Polyethylenimine-modified graphene quantum dots promote endothelial cell proliferation

Endothelial cell proliferation plays an important role in angiogenesis and treatment of related diseases.The aim of this study was to evaluate the effect of polyethylenimine(PEI)-modified graphene quantum dots(GQDs)gene vectors on endothelial cell prolifera-tion.The GQDs-cationic polymer gene vectors were synthesized by amidation reaction,and used to deliver pzNF580 gene to Human umbilical vein endothelial cells(HUVECs)for promoting their proliferation.The chemical modification of GQDs can ad-just gene vectors'surface properties and charge distribution,thereby enhancing their interaction with gene molecules,which could effectively compress the pZNF580 gene.The CCK-8 assay showed that the cell viability was higher than 80%at higher vector concentration(40μg/mL),demonstrating that the GQDs-cationic polymer gene vectors and their gene complex nanoparticles(NPs)having low cytotoxicity.The results of the live/dead cell double staining assay were consistent with those of the CCK-8 assay,in which the cell viability of the A-GQDs/pZNF580(94.38±6.39%),C-GQDs-PEI-polylactic acid-co-polyacetic acid(PLGA)/pZNF580(98.65±6.60%)and N-GQDs-PEI-PLGA/pZNF580(90.08±1.60%)groups was significantly higher than that of the Lipofectamine 2000/pzNF580(71.98±3.53%)positive treatment group.The results of transfection and western blot experiments showed that the vector significantly enhanced the delivery of plasmid to HUVECs and increased the expression of pZNF580 in HUVECs.In addition,the gene NPs better promote endothelial cell migration and proliferation.The cell migration rate and proliferation ability of C-GQDs-PEI-PLGA/pZNF580 and N-GQDs-PEI-PLGA/pZNF580 treatment groupS were higher than those of Lipofectamine 2000/pDNA treatment group.Modified GQDs possess the potential to serve as efficient gene carriers.They tightly bind gene molecules through charge and other non-covalent interactions,significantly improving the effciency of gene delivery and ensuring the smooth release of genes within the cell.This innovative strategy provides a powerful means to promote endothelial cell proliferation.