The Expression Of Antimicrobial Peptide AWRK6 In Bacillus Subtilis And The Effect On The Growth Of Chicken

Antibacterial peptide(ABP),also called antimicrobial peptide(AMP)or peptide antibiotics,is expressed by immune defense systems of all kinds of organism and it is a kind of small molecule peptides that possess immunomodulatory activity and broad-spectrum antimicrobial activity,it is also a important component of non-specific defense system and exists in almost all forms of life such as plants,insects,amphibians,fish,birds,mammals including the human body.Antimicrobial peptides typically contain 12 to 50 amino acids,and the relative molecular mass is in the range of 2kDa?8kDa.Researches find that the antimicrobial peptides have broad-spectrum antimicrobial activity,they envelop virus and tumor cell through the chemotactic response on dendritic cells,monocytes and memory T cells,it becomes an important medium between the innate and acquired immune responses,starting the acquired immune system to improve the body's resistance to microbial infection.Dybowskin-2CDYa(SAVGRHGRRFGLRKHRKH)is a new natural antibacterial peptide which is found and named by our laboratory in frog skin,after being analysed and designed by bioinformatics and then transformed,we've got a more efficient and stable antimicrobial peptides AWRK6(SWVGKHGKKFGLKKHKKH),the transformed peptide AWRK6 consists of 18 amino acids,the molecular weight is 2130.5,the structure mainly is alpha-helix,and has an isoelectric point of 9.60.According to the sequence of peptide AWRK6,by the means of Touchdown Booster PCR method we got full-length gene of AWRK6 and it could be inserted into the E.coli-Bacillus suhtilis shuttle vector,and being expressed in the protease-deficient Bacillus subtilis WB600.Through PCR method,a protein tag named Glutathione S-transferase enzyme(GST)is obtained,the promoter of Levansucrase and signal peptide sequence(SacB p.s.,SacR)is prepared to be the expression regulatory element,after connecting and transfering them into plasmid pHY300-plk,a shuttle vector pHYSGA is obtained,then it's transformed into Bacillus subtilis WB600,we verified the result using PCR and double enzyme digestion method.Then induced it to express secretory antibacterial peptide AWRK6,we identified the product is correctly expressed and folded with SDS-PAGE and Western-Blot examination.Aiming at the medium,we tested the antimicrobial effect and the influence on the growth performance of chicken as a kind of feed additive.Conclusion:By constructing a inducible expression system of Bacillus subtilis and expressing antimicrobial peptides AWRK6,the product has antibacterial activity for E.coli,and Staphylococcus aureus.Add thallus to the chicken's feed and we find that the growth of chicks' ratio of meat can be effectively improved,meantime,we ran the ELISA test on the chicken serum immune parameters,found that the addition of peptide AWRK6 significantly improved the content of the immune factors,and laid the foundation for the follow-up research on the Bacillus subtilis antimicrobial peptide expression system feed applications.