The Study On Cloning And Targeted Modification Of Antibacterial Peptide Hepcidin From Fish

Due to the overuse and abuse of traditional antibiotics,bacterial resistance causes serious health problems worldwide,making it difficult to find new and effective antimicrobial drugs.Antimicrobial peptides(AMPs),as both antimicrobial agents and important regulatory factors of the natural immune system,have undoubtedly become a promising antimicrobial agent against a variety of pathogenic microorganisms.Hepcidin is an antimicrobial peptide and iron regulating hormone,which is mainly produced by liver and released into serum.It plays a dual role in host innate immunity and iron metabolism.Hepcidin is of a wide range of biological activities against both Gram-negative bacteria and Gram-positive bacteria,and also of antiviral,anti-parasitic and anti-fungal growth inhibition activity.However,natural antimicrobial peptides are of poor stability,short half-life,strong toxic and side effects,and especially the possibility of severe hemolytic activity.This experiment mainly carried out the cloning of fish antibacterial peptide Hepcidin gene,bioinformatics analysis,sequence-template-method to optimize the design of antimicrobial peptide and the expression of antimicrobial peptide in Pichia pastoris,including the following four parts:The first part is the cloning of the antimicrobial peptide gene Hepcidin in Carassius auratus gibelio.Based on the results of the amino acid sequence alignment of Hepcidins,degenerate primer were designed.The total RNA of Carassius auratus gibelio tissue was extracted,and c DNA was synthesized by reverse transcription;after PCR amplification,it was cloned into T vector and transformed into competent E.coli DH5α for culture.After colony PCR and sequencing identification,the antimicrobial peptide Hepcidin was successfully cloned.Through NCBI Blast multiple sequence homology comparison,it was verified that its sequence belongs to the fish Hepcidin antimicrobial peptide,and similarity with that of zebrafish is 100%.The second part is bioinformatics analysis of the antimicrobial peptide Hepcidin.The phylogenetic analysis of Hepcidin gene family showed that the phylogenetic tree of Hepcidin was mainly divided into three branches.The longest evolutionary relationship between zebrafish and Clupea harengus was 46.15%.It had the highest similarity to Labeo rohita Hepcidin,accounting for 72.22%.Hepcidin was analyzed by software such as Ex PASy,Inter Pro,TMHMM2.0,TMpred,Signal P-5.0 Server and Phyre.It was found that the fragment 1-24 was the signal peptide,25-64 was the propeptide,and 67-91 was the active peptide.The signal peptide is located in the N terminal of Hepcidin antimicrobial peptide and of hydrophobicity.In the transmembrane region,the direction of the transmembrane helix is from inside to outside,and the helix is formed by amino acids at position 6-28,and the central site is amino acid at position 18.The N-terminal of Hepcidin is mainly α-helix,and the Cterminal is mainly β-fold.The confidence of tertiary structure prediction of mature peptide was 98.6%,and the consistency was 70%.The third part is to establish the sequence template of antimicrobial peptide Hepcidin.Hepcidin peptide related sequences were retrieved by Gen Bank,and fish Hepcidin polypeptide sequences were collected.Meanwhile,domain sequences were determined by comparing and analyzing the natural Hepcidin peptides.The frequency of each amino acid at each position in the domain sequence was calculated,and the sequence template was optimized and sorted out.The anti-cancer index,hemolytic activity,half-life,cell penetration and other parameters of the peptide sequence were optimized by using the obtained sequence template as motif.Antibacterial activity of the template sequence was predicted.It showed that it is of anti-Klebsiella pneumonia and anti-fungal activity,and toxicity to Human erythrocytes.The optimized design of polypeptide Hepcidin-M2 R was of the best antibacterial effect,which could reduce the OD value by 93.5%.The template-sequence-peptide can effectively reduce the OD value of bacterial fluid by 67.9%,and the natural antimicrobial peptide Hepcidin can effectively reduce the OD value of bacterial fluid by 72.2%.SPSS analysis showed that P<0.01 indicating a significant difference,and the optimized design of polypeptide was of obvious bacteriostatic effect.The fourth part is the inducted expression of Hepcidin in Pichia pastoris.The gene coding sequence of the Carassius auratus gibelio antimicrobial peptide Hepcidin was optimized by analyzing and designing the preferred codon sequence of Pichia pastoris using online analysis software Codon Usage Database.The optimized and original sequence AMP coding sequence was inserted into the Pichia pastoris expression vector p PIC9 K,and ransformed into KM71,through defective and resistance screening,highcopy transformants are selected,methanol induced expression to obtain recombinant antimicrobial peptides.By comparing the antibacterial effect of the optimized sequence and original sequence through the OD value measured by the antibacterial experiment,it was found that the antibacterial activity of the optimized sequence was higher than that of original sequence,the bacterial fermentation supernatant was 27% lower than that of original sequence.The higher the concentration of G418,the stronger the antibacterial activity of the screened transformants.In this study,the Carassius auratus gibelio antibacterial peptide Hepcidin gene was cloned,its structures and properties were analyzed by utilizing bioinformatics technology.The innovation point was to optimize and mutate the antimicrobial peptide by using sequence template method and amino acid mutation method.The experimental results showed that the antibacterial activity of the optimized antibacterial peptide was enhanced.This study is expected to provide technical support for the targeted modification of fish antimicrobial peptides and application in breeding for disease prevention and treatment.