人抗氧化蛋白Peroxiredoxin 1和2的表达、纯化及功能研究

发布时间：2018-04-15 03:00

  本文选题：抗氧化蛋白 + 大肠杆菌　； 参考：《华南理工大学》2012年硕士论文

【摘要】：人抗氧化蛋白Peroxiredoxin1（Prx1）和Peroxiredoxin2（Prx2）是人体内重要的过氧化物酶，具有清除过氧化物、保护机体免受氧化损伤和参与信号传导等重要生理功能。蛋白组学研究证实Prx1在淋巴瘤，肉瘤，乳腺癌等疾病患者中表达上调；Prx2则在骨肉瘤、阿兹海默症和宫颈癌等疾病患者体内有高表达情况，这表明Prx1和Prx2在疾病诊断治疗方面可能具有重要意义。 本文成功构建了pET28-Prx1和pET28-Prx2原核表达载体，实现了重组质粒在大肠杆菌BL21（DE3）的表达，SDS-PAGE结果显示重组蛋白主要为上清表达，且分子量均与预期大小（~24kD）一致。重组表达后的蛋白经Ni-NTA螯合亲和层析后产量分别达到了7.59mg/g菌体和8.82mg/g菌体，纯度分别为88.5%和91.5%。Western Blotting结果显示纯化后的蛋白带有组氨酸标签，说明成功实现了人Prx1和Prx2在大肠杆菌中的重组表达。 纯化后的Prx1和Prx2在37℃和pH7.4的条件下催化H2O2反应的米氏常数Km分别为2.06×10-4M和1.56×10-4M。质粒保护实验表明Prx1和Prx2可以保护pUC18质粒遭氧化损伤由超螺旋变成缺刻状态。通过从细胞形态学，凋亡检测和ROS检测结果证实紫外照射会引起细胞内ROS含量上升，过量的ROS则会导致细胞凋亡；Prx2的加入可以在一定程度上保护细胞免受紫外引起的氧化损伤，，Prx1的保护效果则不明显。本文对Prx1和Prx2的抗氧化性质的研究为其可能在食品防氧化、化妆品工业和部分疾病的诊断治疗等领域的应用奠定了基础。
[Abstract]:Human antioxidant proteins peroxiredoxin 1 (Prx1) and peroxiredoxin2Prx2 (peroxiredoxin2Prx2) are important peroxidase in human body, which have important physiological functions such as scavenging peroxide, protecting body from oxidative damage and participating in signal transduction.Proteomic studies have confirmed that the up-regulated expression of Prx1 in patients with lymphoma, sarcoma, breast cancer and other diseases is highly expressed in patients with osteosarcoma, Alzheimer's disease and cervical cancer.This suggests that Prx1 and Prx2 may play an important role in the diagnosis and treatment of disease.In this paper, prokaryotic expression vectors of pET28-Prx1 and pET28-Prx2 were successfully constructed, and the SDS-PAGE results showed that the recombinant protein was mainly expressed in supernatant, and its molecular weight was consistent with the expected size of 24kD.The recombinant protein was chelated by Ni-NTA affinity chromatography to produce 7.59mg/g and 8.82mg/g, respectively. The purity of the purified protein was 88.5% and 91.5%.Western Blotting showed that the purified protein was labeled with histidine.The recombinant expression of human Prx1 and Prx2 in Escherichia coli was successfully realized.The Michaelis constants km of H2O2 reaction catalyzed by purified Prx1 and Prx2 at 37 鈩

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