Expression,Purification And Activity Of Two Antimicrobial Peptide Mytichitin-CB-Hispidalin In Chlamydomonas Reinhardtii

The discovery of antibiotics has made a tremendous contribution to the medical industry,and it has also caused a series of drug resistance problems.At this time,the emergence of antimicrobial peptides has caused people's attention.Mytichitin-CB antimicrobial peptides have no inhibitory effect on Gram-negative bacteria.Hipidalin antimicrobial peptides are sensitive to pepsin and papain.After literature reading,it is found that the recombinant expression of different antimicrobial peptides can improve their antibacterial activity and stability.In this project,the single-cell eukaryote of Chlamydomonas reinhardtii was used as a cell factory to construct a Mytichitin-CB-Hispidalin hybrid antibacterial peptide recombinant expression vector,and to try to obtain a new antibacterial with high antibacterial activity and high stability Peptide.In this experiment,antibacterial peptides were screened on selective medium containing bleomycin,and 7 transgenic strains stably expressing Mytichitin-CB-Hispidalin hybrid were obtained.Through Ni-NTA affinity purification and gel filtration purification,the target protein was enriched at the expected molecular weight of 17 kDa,and the yield was approximately 0.18%of the total soluble protein.Through MIC and bacteriostatic circle experiments,5 Gram-negative and Gram-positive strains were tested for antibacterial activity.The area of Mytichitin-CB-Hispidalin hybrid antibacterial peptides at 1×MIC concentration was 50 ?g/156%-208%of mL gentamicin(positive control)shows strong antibacterial efficacy.Using Salmonella enteritidis(ATCC 10467)as the test bacteria,Mytichitin-CB-Hispidalin hybrid antimicrobial peptides were tested for stability under different temperature,pH and protease conditions.The results showed that it was at a wider temperature It can maintain strong antibacterial activity in the range of pH and pH,and has strong stability to all tested proteolytic enzymes.At the same time,at a concentration of 320 ?g/mL Mytichitin-CB-Hispidalin hybrid antimicrobial peptide,the hemolysis in rat red blood cells was 6.3%,and 480 ?g/mL Mytichitin-CB-Hispidalin hybrid antimicrobial peptide against HEK293 and MDBK cells Cytotoxicity is low.In summary,Chlamydomonas reinhardtii can be used as a cell factory to produce Mytichitin-CB-Hispidalin antibacterial peptides with stable antibacterial activity,which can be used in food processing and storage fields.As a food-grade organism,Chlamydomonas reinhardtii can be added to animal feed,eliminating the purification process,which will benefit the development of green animal husbandry industry.