Cloning, sited-directed mutagenesis and expresison of hCu, Zn-SOD gene in E.coli

  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.