TAT-Cytoglobin融合蛋白的原核表达、纯化及生物活性分析  

Expression,purification,and characterization of fusion protein TAT-Cytoglobin

作　　者：张如婧 李招发[1,2] 施伟杰[1] 许瑞安[1,2] 

机构地区：[1]华侨大学生物医学学院,福建泉州362021 [2]分子药物教育部工程研究中心,福建泉州362021

出　　处：《生物工程学报》2014年第8期1247-1255,共9页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(Nos.81072578;81271692)资助~~

摘　　要：旨在利用细胞穿膜肽TAT的穿膜作用和细胞珠蛋白(Cytoglobin,Cygb)的抗衰老、抗纤维化的功能,将二者通过基因工程的手段融合在一起,以期获得能够穿透细胞屏障的Cygb。通过两次重叠PCR技术获得了TAT-Cygb DNA,将其插入原核表达载体pET22b质粒中,转化至大肠杆菌BL-21,筛选出可表达TAT-Cygb融合蛋白的大肠杆菌工程菌株。经乳糖诱导表达TAT-Cygb,CM阳离子交换层析(CM Sepharose Fast Flow Protocol)获得纯度高达95%的TAT-Cygb融合蛋白,分子量约23 kDa。生物活性实验显示,TAT-Cygb过氧化物酶比活力达到(422.30±0.36)U/mg。TAT-Cygb预处理的Hacat细胞可免受H2O2氧化应激的损伤(RGR=100%),同时TAT-Cygb可治疗已被H2O2氧化损伤的细胞(RGR=98%),与Cygb处理组相比具有显著差异(RGR=79%)。该研究首次成功利用大肠杆菌表达系统表达了可穿透细胞膜的、有生物活性的TAT-Cygb融合蛋白,为继续开展Cygb在抗衰老、抗纤维化和抗癌领域的研究奠定了基础。The aim of this study was to obtain a cell-penetrating cytoglobin （Cygb）, which combines the transmembrane function of cell-penetrating peptides TAT with the anti-aging and anti-fibrotic role of cytoglobin. The Cygb gene was complexed with TAT gene by overlapping PCR, inserted into the vector pET22b to construct the recombinant expression plasmid （pET22b-TAT-Cygb） and then transformed into Escherichia coli BL21 （DE3）. The fusion protein TAT-Cygb, whose expression was induced by lactose, was purified by CM Sepharose Fast Flow Protocol and verified by Western blotting. The final TAT-Cygb had a molecular weight of 23 kDa with 95% purity, as shown by SDS-PAGE. As demonstrated by bioactivity experiments, TAT-Cygb exhibited a high specific peroxidase activity up to （422.30±0.36） U/mg. Both TAT-Cygb and Cygb pretreatment group could protect Hacat cells against oxidation of H202, but only TAT-Cygb treatment group could remedy cells injuried by H202 （RGR=98%）, which was significantly different from Cygb treatment group （RGR=79%）. We successfully obtained the bioactive and cell-penetrating fusion protein TAT-Cygb that has the potential application in anti-aging, anti-fibrotic and anti-cancer.

关 键 词：细胞穿膜肽 细胞珠蛋白 融合蛋白 纯化 氧化应激 

分 类 号：R3411[医药卫生—基础医学]