Cloning And Expression Of The Hybrid Antimicrobial Peptide Sα-Jp Gene

Hybrid antimicrobial peptide is a new type of antibacterial peptides, which using genetic engineering method to connect different antimicrobial peptide fragments together. Build a new kind of hybrid antimicrobial peptides is an important method to transform the natural antimicrobial peptides to optimize. Hybrid antimicrobial peptides overcome the defect of natural antibacterial peptide and integrate the merits of different antibacterial peptides. Hybrid antimicrobial peptide is a hotspot of current research.Based on the genetic engineering and molecular biology technology, the artificially synthesized hybrid antimicrobial peptide Sα-Jp gene was cloned and expressed in the eukaryotic.Optimize the expression conditions, study the biological activities and properties of the express product after purification,the main results are as follows: 1. The cloning of hybrid antimicrobial peptides Sα-Jp gene:According to the amino acid sequence of Spinigerin a, Japonicin Ⅱ, and the Pichia pastoris preference codon design hybrid antimicrobial peptide Sα-Jp gene sequence and primers; successfully amplification of hybrid antibacterial peptide gene Sα-Jp by the PCR technology. Cloning the target gene to the pPICZαA plasmid and constructing the recombinant expression vector pPICZαA-Sα-Jp. The test results show that the eukaryotic expression vector pPICZαA-Sα-Jp has built successfully. 2. The expression of Sα-Jp gene:Recombinant plasmid was lineared by SacI single enzyme and transformed into host bacterium GS115 by electroporation. We used Zeocin to screen out high copy transformants. The correct positive GS115-Sα-Jp was induced by methanol in BMMY medium. Bradford method is used to detect the expression content of the purpose protein in the supernatant. Results show that the amount of Sα-Jp peaked at 10.7 mg/L after 96 h. We conduct the SDS-PAGE electrophoresis after the Nickel column purification. As the predict, has the 5.0 kD protein band. 3. Optimizing the expression conditions:Through optimizing the expression conditions we obtained the best expression conditions: induction time 96 h, induction temperature 28℃, additional 0.5% methanol concentration per 24 h, the initial pH of culture medium is 6.The quantity of the hybrid antimicrobial peptide Sα-Jp under the best inducing conditions can reach to 13.6 mg/L。 4. The study of biological activity and properties:Hybrid antimicrobial peptide Sα-Jp has better antimicrobial activity on E.coli, staphylococcus aureus, salmonella;and the MIC is 0.100mg/mL 、 0.160mg/mL 、0.140mg/mL.The effect on the E.coli is strong that the diameter of inhibition zone is 23.2 mm. But has no effect on aspergillus Niger and beer yeast. Thermal stability, when 100℃heating 25 min are still active. Better tolerance, are active in pH3-10. The antibacterial activity is still better when in the pH 6-7. With radiation resistant, still active after ultraviolet irradiation for 1h. Genetic stability, the target gene is not lost after passaging 50 times, and the expression product still better antibacterial activity.