【摘要】 目的　在克隆人源铜锌超氧化物歧化酶基因 (hCu ,Zn -SOD)的基础上 ,对hCu ,Zn -SOD进行定点突变 ,使其在E .coliDH5α中表达 ,为进一步改造hCu ,Zn -SOD基因奠定基础。方法　首先构建质粒 pESOD ,然后用定点突变技术把其中hCu ,Zn -SOD的Cys111密码子突变为Ala111密码子 ,再与 pUCMT1相连接 ,构建表达载体pE SODT111,使其在E .coliDH5α中表达 ,表达产物用改良的邻苯三酚法和Westem杂交测定。结果　hCu ,Zn -SOD突变后在E .coliDH5α中成功表达 ,表达产物具有SOD活性 ,活力为 16 4 4 7U/ml培养液。结论　hCu ,Zn -SOD基因经过定点突变后构建的表达载体可在DH5n中表达 ,表达产物同样具有天然的hCu ,Zn -SOD活性更多还原

【Abstract】 Objective For further researching of hCu,Zn-SOD gene engineering,to mutate human copper,zinc-superoxide dismutage gene(hCu,Zn-SOD),and expressed the plamid in E.coli DH5α.Methods The pESOD plamid was constructed firstly,then the Cys 111 genetic code of hCu,Zn-SOD gene in the pESOD plamid was mutated into the Ala 111 code with sited directed mutagenesis and the expression plamid pESODT111 was constructed by inserting the mutated pESOD into pUCMT1 plamid.The pESODT111 plamid was expressed in E.coli JM101.The expression product was determined by Western blot and improved by pyrogallol autoxidation. Results Mutated hCu,Zn-SOD gene expressed in E.coli DH5α correctly,the expression product had SOD activity-16.447?U/ml culture medium.Conclusion The expression product of the mutated hCu,Zn-SOD had activity of native hCu,Zn-SOD.更多还原