【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。更多还原

【Abstract】 Objective To construct prokaryotic expression vector pET15b-PEP-1-SOD1 and express and purify PEP-1-SOD1 fusion protein.Methods The SOD1 cDNA was amplified by PCR using plasmid pBluscript Ⅱ SK-SOD1 as a template,identified by restriction analysis and used for construction of prokaryotic expression vectors pET15b-SOD1 and pET15b-PEP-1-SOD1.The constructed recombinant plasmids were identified by sequencing and transformed to E.coli BL21(DE3) for expression of SOD1 protein and PEP-1-SOD1 fusion protein.The expressed products were identified by SDS-PAGE and Western blot.Results SOD1 and PEP-1-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-1-SOD1 fusion protein were successfully prepared.更多还原