原核表达的萝卜PHGPx对黑色素瘤B16细胞紫外辐射损伤的恢复作用  

Effects of Prokaryotic Expressed Radish Phospholipid Hydroperoxide Glutathione Peroxidase on Restoration of B16 Melanoma Cells Irradiated with Ultroviolet

作　　者：李甜[1] 刘冠兰[1] 段明星[1] 余世实[1] 刘进元[1] 严泽民[2] 徐之伟[2] 

机构地区：[1]清华大学生命科学学院,北京100084 [2]江苏隆力奇生物科技股份有限公司,常熟215555

出　　处：《中国生物工程杂志》2010年第2期66-70,共5页China Biotechnology

基　　金：国家"863"计划(2007AA100604);国家自然科学基金(30170080;39770078);国家"973"计划(2006CB101706)资助项目

摘　　要：将大肠杆菌中高效表达的萝卜PHGPx(RsPHGPx)和突变的RsPHGPx(mRsPHGPx)融合蛋白,经Glutathione Sepharose 4B柱亲和纯化和PreScission蛋白酶柱上酶切,获得了不含GST标签的RsPHGPx和mRsPHGPx重组蛋白,纯度在90%以上。酶活测定验证了活性中心Cys突变的mRsPHGPx的活性显著低于RsPHGPx。考察RsPHGPx和mRsPHGPx对紫外辐射损伤后的黑色素瘤B16细胞的恢复作用,发现活性较高的RsPHGPx能有效提高细胞存活率,降低细胞膜脂质过氧化水平,而活性极低的mRsPHGPx则几乎没有效果,提示由于RsPHGPx能快速清除多种脂类过氧化物,可能主要通过抑制紫外辐射造成的细胞膜脂质过氧化损伤起作用。The radish phospholipid hydroperoxide glutathione peroxidase（RsPHGPx） and its mutant（mRsPHGPx） fusion proteins expressed in Escherichia coli BL21（DE3） with high efficiency were purified with Glutathione Sepharose 4B affinity chromatography and cleaved on-column with PreScission protease,and both the recombinant proteins without tags were obtained at a purity more than 90%.The enzymatic measurement confirmed that the activity of mRsPHGPx was far less than that of RsPHGPx.Effects of RsPHGPx and mRsPHGPx on restoration of B16 melanoma cells irradiated with ultroviolet were investigated.The results showed that the active RsPHGPx could significantly increase the cell viability and reduce the lipid peroxidation of cell membranes,while mRsPHGPx with very low activity had no effects,suggesting RsPHGPx could inhibit the ultroviolet-mediated injury in cell membranes mainly by rapidly scavenging various lipid peroxides.

关 键 词：萝卜PHGPx 定点突变 紫外辐射 细胞恢复 

分 类 号：Q789[生物学—分子生物学]