PEP-1-SOD1融合蛋白的表达及纯化  

Expression and Purification of PEP-1-SOD1 Fusion Protein

作　　者：丁鹏[1] 王家宁[2] 杨波[1] 黄永章[2] 骆丽娜[3] 

机构地区：[1]武汉大学医学院附属人民医院,武汉430060 [2]郧阳医学院附属人民医院临床医学研究所,十堰442000 [3]河南省平顶山煤业集团总医院,平顶山467000

出　　处：《中国生物制品学杂志》2006年第3期240-243,共4页Chinese Journal of Biologicals

摘　　要：目的构建原核表达载体pET15bPEP1SOD1,并进行PEP1SOD1融合蛋白表达和纯化。方法以pBluescriptIISKSOD1质粒为模板,PCR扩增SOD1cDNA。经酶切后,分别构建原核表达载体pET15bSOD1和pET15bPEP1SOD1,经测序证实构建成功后,分别转化E.coliBL21(DE3),表达SOD1和PEP1SOD1融合蛋白,并进行鉴定。结果SDSPAGE和Westernblot分析表明,分别在相对分子质量22000和26000处出现SOD1和PEP1SOD1目标表达条带,目的蛋白占菌体总蛋白的30%,两种表达蛋白以天然的、可溶性的形式存在。结论已成功地制备出SOD1和PEP1SOD1融合蛋白。Objective To construct prokaryotic expression vector pET15b-PEP-1-SOD1 and express and purify PEP-I-SOD1 fusion protein. Methods The SOD1 cDNA was amplified by PCR using plasmid pBluseript Ⅱ SK-SOD1 as a template, identified by restriction analysis and used for construction of prokaryotie expression vectors pET15b-SOD1 and pET15b-PEP-1-SOD1. The construeted recombinant plasmids were identified by sequencing and transformed to E. coli BL21 （ DE3 ） for expression of SOD1 protein and PEP-I-SOD1 fusion protein. The expressed products were identified by SDS-PAGE and Western blot. Results SOD1 and PEP-I-SOD1 proteins, with relative molecular weights of 22 000 and 26 000 respectively, were expressed. The expressed product existed in a nature and soluble form and contained 30% of total somatic protein. Conclusion SOD1 protein and PEP-I-SOD1 fusion protein were successfully prepared.

关 键 词：铜 锌-超氧化物歧化酶 原核表达 TA克隆 PEP-1肽 融合蛋白 

分 类 号：R996.3[医药卫生—毒理学] R392.12[医药卫生—药学]