Ginseng Of Cu/Zn-SOD Prokaryotic Expression Vector, Expression And Puriifcation

Objective: The build Pet28(a)-Cu/Zn-SOD prokaryotic expression vector,and theestablishment of ginseng Cu/Zn-SOD expression, purification process,the theoretical basisfor the subsequent development and application.Method:1Application of improved Trizol extraction of ginseng leaf total RNA, using molecularcloning method, amplified via RT-PCR ginseng leaf Cu/Zn-OD coding gene, constructingPet28(a)-Cu/Zn-SOD expression vector.2Will to contain Pet28(a)-Cu/Zn SOD Recombinant plasmid was transformed into E.coli BL21(DE3) competent cells, Containing recombinants strains was induced by IPTG,preferred optimal induction of expression conditions,and purification of washing theinclusion body。3The use of nickel ions (Ni+) affinity chromatography purification of the target proteinand the establishment of a purification process。4The xanthine oxidase method for the determination of target protein enzyme activity.。Result1RT-PCR amplification of Cu/Zn-SOD gene sequences in GenBank database publishedin the ginseng of Cu/Zn-SOD gene sequence homology of99%.2Contain Pet28(a)-Cu/Zn-SOD recombinants IPTG induction, the optimized inductiontime of3h, by adding the inducer IPTG final concentration of1mM, the target proteinexpression level was about44.42%.3The molecular weight of the target protein for1.63KD yield of about26.02%.4After the purification of ginseng Cu/Zn-SOD protease activity reached10596.6970U/mg。Conclusion:With the method of molecular biology, building on the success of the ginseng Pet28(a)-Cu/Zn-SOD prokaryotic expression vector, and high expression amount, purificationprocess is simple, convenient mass production, as the next step to study Cu/Zn-SOD biologyfunction of the foundation.。...