Construction Of Temperature Control Prokrytic Expression Vector, Expression And Purification Of Panax Ginseng C.A. Mey Superoxide Dismutase

Objective: In this research, the gene of Cu/ZnSOD of Panax ginseng C.A. was cloned into an prokaryocyte expression plasmid and Cu/ZnSOD inclusion bodies was expressed in E.coli in high efficiency. Then the inclusion bodies were purified by ion-exchange chromatography and molecular sieve chromatography. These results are to provide a foundation for further studies on the radioresistant mechanism of Panax ginseng C.A. and for further development and application of Cu/ZnSOD.Methods:1.Cu/ZnSOD gene was amplified by PCR from genomic RNA of Panax ginseng C.A. and inserted into expression plasmid plasmid pBV220 to construct pBV220-Cu/ZnSOD. The recombinant plasmid was transformed into cloning host E.coli DH5α. The positive clones were selected by using Ampicillin resistance, and recombinant plasmid was identified by enzyme digestion and sequence analysis.2.The recombinant plasmids were transformed to E.coli DH5αand pBV220-Cu/ZnSOD expression in the form of inclusion bodies were gained after induction with temperature. Select the best expression conditions.3.Then the inclusion bodies were purified by ion-exchange chromatography and molecular sieve chromatography.4.Study on the enzyme activity of the purified Cu/ZnSOD fusion protein.Results:1. The recombinant expression plasmid pBV220-Cu/ZnSOD was constructed. Sequence analysis indicated that the target Cu/ZnSOD gene was 459bp, coding 152 amino acid residues. There is a one-base difference between the obtained sequence and the previously published sequence GenBank, the homology was 99%.2. The recombinant plasmids were transformed to E.coli. DH5αand pBV220-Cu/ZnSOD expression in the form of inclusion bodies were gained after induction with temperature.3. The target protein was found after SDS-PAGE, accordant with anticipation. After purified by chromatography, the Cu/ZnSOD protein showed almost a single band after SDS-PAGE.4. The purified Cu/ZnSOD protein specific activity reaches 9389.96U/mg. Conclusion:The recombinant expression plasmid pBV220- Cu/ZnSOD was constructed successfully. The Cu/ZnSOD protein was expressed effectively and found by SDS-PAGE. By affinity chromatography, the purified product could be obtained with a high purity, high enzyme activity. These results will provide theoretical and experimental foundation for further studies on the radioresistant mechanism of Panax ginseng C.A. Mey and on establishing a convenient and efficient way to product Cu/ZnSOD. This will be helpful for the development and application of Cu/ZnSOD.