本文選題：抗氧化蛋白 + 大腸桿菌　； 參考：《華南理工大學(xué)》2012年碩士論文

【摘要】：人抗氧化蛋白Peroxiredoxin1（Prx1）和Peroxiredoxin2（Prx2）是人體內(nèi)重要的過氧化物酶，具有清除過氧化物、保護機體免受氧化損傷和參與信號傳導(dǎo)等重要生理功能。蛋白組學(xué)研究證實Prx1在淋巴瘤，肉瘤，乳腺癌等疾病患者中表達上調(diào)；Prx2則在骨肉瘤、阿茲海默癥和宮頸癌等疾病患者體內(nèi)有高表達情況，這表明Prx1和Prx2在疾病診斷治療方面可能具有重要意義。 本文成功構(gòu)建了pET28-Prx1和pET28-Prx2原核表達載體，實現(xiàn)了重組質(zhì)粒在大腸桿菌BL21（DE3）的表達，SDS-PAGE結(jié)果顯示重組蛋白主要為上清表達，且分子量均與預(yù)期大小（~24kD）一致。重組表達后的蛋白經(jīng)Ni-NTA螯合親和層析后產(chǎn)量分別達到了7.59mg/g菌體和8.82mg/g菌體，純度分別為88.5%和91.5%。Western Blotting結(jié)果顯示純化后的蛋白帶有組氨酸標(biāo)簽，說明成功實現(xiàn)了人Prx1和Prx2在大腸桿菌中的重組表達。 純化后的Prx1和Prx2在37℃和pH7.4的條件下催化H2O2反應(yīng)的米氏常數(shù)Km分別為2.06×10-4M和1.56×10-4M。質(zhì)粒保護實驗表明Prx1和Prx2可以保護pUC18質(zhì)粒遭氧化損傷由超螺旋變成缺刻狀態(tài)。通過從細胞形態(tài)學(xué)，凋亡檢測和ROS檢測結(jié)果證實紫外照射會引起細胞內(nèi)ROS含量上升，過量的ROS則會導(dǎo)致細胞凋亡；Prx2的加入可以在一定程度上保護細胞免受紫外引起的氧化損傷，，Prx1的保護效果則不明顯。本文對Prx1和Prx2的抗氧化性質(zhì)的研究為其可能在食品防氧化、化妝品工業(yè)和部分疾病的診斷治療等領(lǐng)域的應(yīng)用奠定了基礎(chǔ)。
[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

本文編號：1752191