Cloning, Expression, Purification And Enzyme Activity Analysis Of Panax Ginseng And Homo Sapiens Cu/Zn-SOD

Superoxide dismutase (SOD), which can protect cells from damage by reactive oxygen species, is a kind of common antioxidant enzymes.Objective:The purpose of this study is to get two recombinant plasmids by connecting Panax ginseng Cu/Zn-SOD (pCu/Zn-SOD) and Homo sapiens Cu/Zn-SOD (hCu/Zn-SOD) genes with vector pET-22b, express and purify SOD, analyze their activity and obtain SOD of high expression and high activity for the detection and control of animal diseases.Methods:(1) The total RNA were extracted from Panax ginseng and mesenchymal stem cell (MSC), the target genes were obtained by RT-PCR, and were connected to prokaryotic expression vector of vetor pET-22b, and the correctness were verified by PCR and enzyme digestion, then sequencing was performed. (2) The recombinant plasmids were transformed into Ecoli BL21 bacteria for protein expression, the culture were collected, centrifuged and the protein was purified by superdex75 molecular chromatography and DEAE Fast Flow ion exchange chromatography. (3) Bradford method was used to determine the protein concentrations, and Pyrogallol Self-Oxidation method was used to determine the enzyme activity. (4) The theoretical model of pCu/Zn-SOD was built using SWISS-Model, and was compared with the three-dimensional structure of hCu/Zn-SOD. (5) The influnces of temperature and pH on enzymatic activity were also discussed.Results:(1) Recombinant plasmids were successfully constructed. The sequence of target gene conneced into vector is right, the homology between target gene and sequence from GeneBank are 99% and 100%, respectively. (2) The recombinant plasmids were transformed into Ecoli BL21 bacteria, and were induced and expressed, the target protein was found in supernatant, a single protein band was isolated by SDS-PAGE. (3) Cu/Zn-SOD of high activity were obtained, the pCu/Zn-SOD enzyme activity was 13184.4927U/mg, while hCu/Zn-SOD enzyme activity was 11279.2361U/mg. This two enzymes showed a relatively high stability under different temperature and pH.Conclusion:Panax ginseng Cu/Zn-SOD and Homo sapiens Cu/Zn-SOD could be successfully expressed in prokaryotic expression system. The enzyme activity of soluble CuZn-SOD obtained in this study was higher than that of Cu/Zn-SOD expressed in the form of inclusion bodies or extracted directly from plants, and they were stable under different temperature and pH.