胞红蛋白表达纯化及抗氧化活性研究  

Study on Purification and Antioxidant Activity of Recombinant Human Cytoglobin (rhCYGB)

作　　者：接振旺[1] 贾晓健[1] 钱杰[1] 吴梧桐[1] 张玉彬[1] 

机构地区：[1]中国药科大学生物化学教研室,江苏南京210009

出　　处：《药物生物技术》2010年第1期45-49,共5页Pharmaceutical Biotechnology

基　　金：江苏省2008年度"六大人才高峰"资助项目(No.2008035)

摘　　要：研究人胞红蛋白(Cytoglobin,CYGB)的表达纯化、复性和抗氧化活性。人胞红蛋白基因工程菌pET28a/hC YGB/BL21(DE3)经IPTG诱导表达,包涵体分离,复性,透析和Sephacryl S-200层析分离,获得纯化的rhC YGB。SDS-PAGE和HPLC检测表明rhC YGB纯度达95%以上,紫外光谱分析证明rhC YGB分子含有血红素,圆二色谱分析表明该复性蛋白质具有正确的α-螺旋结构。生化分析及细胞水平试验表明rhC YGB具有过氧化物酶活性和抗氧化活性。小鼠急性乙醇肝损伤模型试验表明rhC YGB能有效保护乙醇所致的肝损伤。研究结果表明经添加血红素复性和纯化所得的rhC YGB具有正确的空间结构和抗氧化活性。To refold rhCYGB effectively from inclusion bodies and to further explore the function of rhCYGB, the genetic engineering cells were cultured under the optimized fermentation conditions. Inclu- sion bodies were isolated from rhCYGB engineering cells, and dissolved in guanidine hydrochloride. Native rhCYGB from apoprotein was reconstructed by adding a 1.4 fold excess of hemin hydroehloride. Then the purified rhCYGB was obtained by dialysis and Sephacryl S200 column chromatography. The yield of purified rhCYGB is 5 mg/L culture, SDS-PAGE and HPLC analysis showed that its purity is above 95%. UV spectra and circular dichroism （CD） spectroscopy indicated that purified rhCYGB contained heme and α-helix secondary structure. The rhCYGB can accelerate the decomposition of hydrogen peroxide in the biochemical experiment, which showed that rhCYGB had peroxidase activity （5 u/mg）. Biological assays showed that rhCYGB had anti-oxidative activity and protecting action against acute ethanol-induced mouse liver injury. The experimental results indicated that purified rhCYGB had correct spatial structure and anti-oxidative activities.

关 键 词：胞红蛋白 肝星状细胞激活相关蛋白 纯化 复性 抗氧化 

分 类 号：Q51[生物学—生物化学]