PEP-1肽介导人过氧化氢酶转导入人脐静脉内皮细胞  

Transduction of Human Catalase Mediated by PEP-1 Peptide into Human Umbilical Vein Endothelial Cells

作　　者：姚玲玲[1] 王家宁[1] 黄永章[1] 陈龙[1] 郭凌郧[1] 孔霞[1] 

机构地区：[1]郧阳医学院附属人民医院临床医学研究所

出　　处：《郧阳医学院学报》2007年第1期9-12,F0003,共5页Journal of Yunyang Medical College

摘　　要：目的:采用体外培养的人脐静脉内皮细胞株(CRL-1730),研究PEP-1肽介导人过氧化氢酶穿透细胞的能力。方法:构建原核表达质粒pET15b-PEP-1-CAT,将其在基因工程菌中表达出N端带有6个组氨酸“标签”(His-tag)的重组蛋白,并通过金属螯合亲和层析对其进行纯化,然后将纯化得到的PEP-1-CAT融合蛋白加入体外培养的人脐静脉内皮细胞,通过Western blot来分析其转导能力。结果:测序分析证实,原核表达质粒pET15b-PEP-1-CAT重组成功。SDS-PAGE和Western blot结果表明,PEP-1-CAT在E.coli中获得了高效表达,经Ni2+-NTA-树脂柱亲和层析纯化得到了融合蛋白PEP-1-CAT,并在体外检测到其具有特异的过氧化氢酶活性,活性值为77.1 U/g。将其加入到体外培养的内皮细胞培养基中,发现它能以剂量依赖及时间依赖的方式转导入细胞内,并能保持酶活性达48 h。结论:PEP-1-CAT融合蛋白能高效地转导入人脐静脉内皮细胞,为防治各种与氧化应激损伤有关的疾病开辟了新的途径。Abstract ： Objective To investigate the transduction ability of PEP - 1 - CAT fusion protein with human umbilical vein endothelial cell （HUVECs） CRL - 1730. Methods Utilizing the TA - cloning program and isocaudamer technique, the pET15b - PEP - 1 - CAT of prokaryotic expression plasmid was constructed. The recombinant plasmid was transformed into E. coli BL21 （DE3） ,and the protein expression was induced by IPTG. The recombinant protein has an N -terminal His- tag which could be used to purify the target protein by affinity chromatography on a Ni^2＋ - NTA - resin column. The fusion protein PEP - 1 - CAT was prepared and characterized by specific enzyme activity in vitro. Then the purified PEP - 1 - CAT fusion protein was added on cultured HUVECs. The tranduction ability of PEP - 1 - CAT fusion protein into cells was analyzed by Western blotting. Results The construction of the recombinant plasmid was proved to be successful in that the PEP - 1 and the human CAT cDNA sequence of pET15b - PEP - 1 - CAT by sequencing were coincided with the designed sequence encoding PEP - 1 peptide and the human catalase cDNA sequence of GeneBank ＂AY028632＂ , respectively. SDS - PAGE and Western blotting demonstrated that PEP - 1 - CAT was expressed and purified successfully, with specific activity 77.1 U/g. The transduction study showed that PEP - 1 - CAT fusion protein could enter endothelial cells in a dose - and time- dependent manner when added exogenously into culture media. Once inside the cells,the transduced PEP- 1 -CAT protein was stable for 48 h. Gonclusion The PEP - 1 - CAT fusion protein can efficiently penetrate the endothelial cells. These findings provide a novel strategy for prevention and therapy of various disorders related to oxidative stress.

关 键 词：过氧化氢酶 PEP-1肽 蛋白转导 脐静脉内皮细胞 

分 类 号：R363[医药卫生—病理学]