Expression,Purification And Bioactivity Of Antimicrobial Peptide ToAmP4 In Chlamydomonas Reinhardtii

The abuse of antibiotics leded to the emergence ofstrains of drug resistance,people are eager to find antibiotics substitution.Antimicrobial peptides with high antibacterial activity、broad antibacterial spectrum and don’t lead to drug resistance,etc.,making antibacterial peptide as a good alternative.Chlamydomonas reinhardtii i can be directly added in animal feed,and there are a lot of mutants,and all the genes have been sequenced,so using Chlamydomonas reinhardtii as a model of biological research has great advantages.In this paper,the antibacterial peptide ToAmP4 from the plant dandelion,showing alkaline,composed of 41 amino acids,the molecule has three disulfide bonds,with broad-spectrum antibacterial,in the prokaryotic expression system successfully expressed and biological activity.The aim of the present study is to express the antimicrobial peptide ToAmP4 in eukaryotic cells of C.reinhardtii,and to study its bioactivity.In order to study the expression of antimicrobial peptides in Chlamydomonas reinhardtii,We must Construct two expression vectors-Ble-FMDV-HA-6His-ToAm P4 and Ble-FMDV-HA-6His-ToAmP4×3.The former is a haploid antimicrobial peptide,which is directly constructed by enzymatic cleavage.The latter is a triploid antimicrobial peptide.The target fragment is obtained by PCR and then digested by restriction enzyme.The constructed expression vectors of Chlamydomonas reinhardtii were transformed into CC125 by electroporation and screened by immunoblotting.The probability of haploid screening was 6.7%,and that of triploid was 10.9%.However,in the haploid algae did not get the cut off the target fragment,while in the triploid antimicrobial peptides are cut off the purpose of the fragment.The content of the target protein in the total protein was determined by ELISA.The results showed that the protein was 0.32%of the total protein.The antimicrobial peptides were purified and purified by nickel-chro-maturation and freeze-dried.The inhibitory rate of MIC was 93.4%when the MIC concentration was 50μg/mL,The inhibition rate of Staphylococcus aureus reached 96.4%.Agar diffusion test(inhibition zone test)in the antimicrobial peptide concentration of 50μg/mL for E.coli O157 inhibition zone diameter of 14mm,the antibacterial peptide concentration of 40μg/mL inhibition of Staphylococcus aureus The diameter is 18mm.In this study,two sets of expression vectors Ble-FMDV-HA-6His-ToAmP4 and Ble-FMDV-HA-6His-ToAmP4×3 were successfully constructed and successfully transferred into the wild type Chlamydomonas reinhardtii,expression.The antimicrobial peptide ToAmP4 has biological activity after being expressed by Chlamydomonas reinhardtii.Chlamydomonas reinhardtii provided a theoretical support for the expression of antimicrobial peptides in eukaryotes as a model of biological expression of antimicrobial peptides.Since Chlamydomonas reinhardtii is a food safety-grade organism,in this study a significant increase in the natural additive to animal feeds Meaning,to improve the expression of the premise,the application of the food alsohas great significance.