超氧化物歧化酶基因突变在大肠埃希菌中表达
Cloning, sited-directed mutagenesis and expresison of hCu, Zn-SOD gene in E.coli
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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pESODT111, 使其在E.coli DH5α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.coli DH5α中成功表达, 表达产物具有SOD活性, 活力为16.447U/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。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 DH5A.Methods The pESOD plamid was constructed firstly, then the Cys111 genetic code of hCu, Zn-SOD gene in the pESOD plamid was mutated into the Ala111 code with sited-directed mutagenesis and the expression plamid pESODT 111 was constructed by inserting the mutated pESOD into pUCMT 1 plamid.The pESODT 111 plamid was expressed in E.coli JM 101.The expression product was determined by Western blot and improved by pyrogallol autoxidation.Results Mutated hCu, Zn-SOD gene expressed in E.coli DH5A 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.
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