Design And Screening Of Novel And High-efficiency Antimicrobial Peptides Based On Cecropins And Their Protective Effect On Ulcerative Colitis Model Mice

AMPs are endogenous peptides expressed constitutively or inducibly by the main mechanism of innate immunity and constitute a fast and efficient organism defense against pathogens.AMPs are widely used because they exist as many species and have high antimicrobial activity,broad antimicrobial spectrum and immunomodulatory functions.The target strain of AMPs are not easy to produce resistance mutations.It can replace antibiotics and can be used as feed additive to regulate the balance of animal intestinal flora and improve the structure of intestinal flora.Cecropins is one of the earliest and most thoroughly studied and effective antimicrobial peptides in the world.Sharks are abundant marine resources,the liver of which accounts for 75% of their internal organs.APSL has immunomodulatory function.Colorectal cancer is the third largest malignancy in the world and is a significant cause of cancer death.The incidence of colorectal cancer in patients with inflammatory bowel disease(IBD)is much higher than in the general population,so the treatment of inflam matory bowel disease is very important.The recombinant protein expressed by APSL gene and Cecropins gene may be an antibacterial peptide with higher activity which could be a new feed additive that can prevent enteritis and become an agent for treating inflammatory bowel disease.The purpose of this project is to use the Cecropins antimicrobial peptide and APSL fusion expression to screen new,efficient antimicrobial peptides,and study its protective effect and mechanism in UC model mouse.In this project,we carried out the bioinformatics analysis to the shark regeneration liver cDNA library and successfully screened a series of gene sequences with a size of 105 bp and an isoelectric point greater than 10.5.By overlapping PCR,16 pET-dute-His-sumo-APSL-Cecropins recombinant plasmids and 10 pET-dute-His-sumoCecropins-APSL recombinant plasmids were constructed.Purified proteins were expressed from the 26 recombinant plasmids.The recombinant APSL863 s protein was found to have higher antibacterial activity at the same concentration.The results showed that the antibacterial activity of recombinant APSL863 s was higher comparing with Cecropin and APSL863 at the same concentration.An antimicrobial peptide with higher antibacterial activity was successfully screened.In this study,we investigated the protective effect and mechanism of the newly screened and efficient antimicrobial peptide(recombinant APSL863s)in UC model mouse.The model of DSS induced enteritis was constructed and the prophylactic treatment was given to the mice.Recombinant APSL863 s protein,cecropin protein and APSL863 protein were administered to mice in each group.The macroscopic changes of body weight,diarrhea,blood in the stool and the large intestine of the mice in each group were observed.Recombinant APSL863 s protein and cecropin protein can reduced the symptoms of colitis in mice(P<0.05),but the remission of recombinant APSL863 s protein was better.The colorectal tissues of each group were HE stained.The normal mouse colon tissue structure is clear.Mucosa and goblet cells are complete.Both recombinant APSL863 s protein and cecropin protein could improve the damage of colonic tissue of mice with enteritis,but the remission of recombinant APSL863 s protein was better.The RNA of the colorectal tissues of each group was extracted and quantitatively measured by qPCR to analyze the changes of the expression of inflammatory cytokines.Recombinant APSL863 s protein could reduce the expression of proinflammatory cytokines such as IFN?,IL1? and IL17?(P<0.05).The intestinal contents of mice(mouse droppings)were sequenced by 16 s rDNA and the results showed that the Firmicutes,Bacteroidestes and Prevotellaceae_UCG-001,Bacteroidetes were closely related to the pathogenicity of DSS-induced mouse enteritis.APSL863 s protein can improve the abundance and diversity of intestinal flora,improve intestinal flora of enteritis mice and regulate the balance of intestinal flora.In this study,we successfully screened a new cecropin-based,highly effective antimicrobial peptide(recombinant APSL863s),which provides protection in DSS-induced mouse enteritis.It can regulate the abundance and diversity of intestinal micro-organisms and improve inflammatory bowel disease.It can be used to treat inflammatory bowel disease.