重组大鼠细胞球蛋白的制备、纯化与活性  

Preparation,Purification and Activity of Recombinant Rat Cytoglobin

作　　者：张亭亭[1] 李招发[1] 许瑞安[1] 

机构地区：[1]华侨大学分子药物学研究所,福建泉州362021

出　　处：《华侨大学学报（自然科学版）》2009年第6期668-672,共5页Journal of Huaqiao University(Natural Science)

基　　金：国家高新技术研究发展(863)计划项目(2005216060;2008AA022135);福建省自然科学基金资助项目(2007J0105);福建省科技厅重点项目(2008Y0084)

摘　　要：通过聚合酶链式反应(PCR)克隆鼠源细胞球蛋白(Cytoglobin,CYGB)基因,构建原核表达载体pET22b-Cygb,转入E.coliBL21(DE3)经乳糖诱导表达CYGB,发现CYGB主要以可溶形式存在,其表达量占可溶性总蛋白的35%以上.通过DEAE阴离子交换柱层析和Sephacryl S-100凝胶过滤层析,CYGB纯度可超过95%.重组表达的可溶性CYGB不但具有过氧化物酶活性,其活性为(3.23±0.12)mkat.(g.min)-1,还能保护胎鼠原代皮肤成纤维细胞减轻氧化应激损伤(H2O2模型).Rat Cygb cDNA was cloned through PCR with rAAV-Cygb as template and inserted into a prokaryotic expression vector to construct a recombinant plasmid, pET22b-Cygb. Then the recombinant plasmid pET22b-Cygb was transformed into E. coli BL21（DE3） and expressed by lactose induction. The CYGB protein accounted for more than 35 % of total soluble protein. The purified CYGB protein was achieved through DEAE anion-exchange chromatography and Sephacryl 8-100 gel filtration chromatography, respectively. Eventually, the purity of CYGB protein was over 95%. After purification, its antioxidation activity was measured through its protection effect to primary skin fibroblast of fetal rat resulted from hydrogen peroxide damage, and its peroxidase activity unit was （3.23±0.12）mkat·（g·min）^-1.

关 键 词：重组细胞球蛋白 高效可溶表达 分离纯化 鉴定 活性检测 

分 类 号：Q512.2[生物学—生物化学] Q503