Screening Of Antimicrobial Peptides Produced By Lactobacillus Casei Stimulated Mice Intestine Cells And Its Effect On Proliferation Of Intestinal Epithelial Cells

Antimicrobial peptides(AMPS)are small molecular peptides with antibacterial and antiviral activities,which generally contain 20-50 amino acid residues,with positive charge and amphiphilic molecular structure.The diversity and anti pathogenic function of AMP make it a hot spot in the research of antimicrobial drugs and preservatives.Some Lactobacilli can stimulate intestinal cells to secrete antimicrobial peptides,remove intestinal pathogens and protect intestinal barrier.Screening antimicrobial peptides secreted by Lactobacillus casei to stimulate intestinal epithelial cells can be used as animal feed additives to effectively resist some intestinal pathogenic microorganisms and protect the integrity of animal intestinal epithelial cells,which has theoretical significance and application value for promoting green and healthy breeding industry.Therefore,this study analyzed the intestinal tissues of mice fed with lactic acid bacteria by high-throughput sequencing technology,screened the high-level expression of antimicrobial peptides,and explored the antibacterial activity,antiviral activity,and cell proliferation and migration promoting ability of antimicrobial peptides expressed in vitro,so as to provide a theoretical basis for the development of broad-spectrum intestinal antimicrobial repair agents.In this study,high-throughput transcriptome sequencing technology was used to analyze the data of intestinal tissues of mice fed with Lactobacillus casei,and the genes with high expression of antimicrobial peptides were screened and verified by q RT-PCR.The selected antimicrobial peptides were expressed in E.coli prokaryotic expression system and Western blot The protein was purified by protein purification system,and then the purified protein was co incubated with enterotoxigenic Escherichia coli(ETEC)reference strains ETEC-K88,ETEC-K99 and ETEC-987 P to verify the inhibitory effect of antibacterial peptide on bacterial or viral proliferation CCK-8 and cell scratch test were used to verify the effect of antibacterial peptide on the proliferation and migration of intestinal epithelial cells;Western blot was used to verify the expression of tight junction related proteins after antibacterial peptide stimulated intestinal epithelial cells,and to detect whether antibacterial peptide stimulated intestinal epithelial cells can activate m TOR and Wnt / β-Catenin upstream signal pathway.Five antimicrobial peptide genes were screened by transcriptome analysis,which were Defa39,Defa35,Defa38,Reg3 a and Reg1.After construction of expression vector and prokaryotic expression,it was found that Defa39,Defa35 and Defa38 could not be expressed;Western blot analysis showed that the band sizes of Reg3 a and Reg1 were consistent with the expected results;the recombinant antimicrobial peptide protein obtained by prokaryotic expression could significantly inhibit ETEC-K88,ETEC-K99 and ETEC-987 P in vitro.The recombinant antimicrobial peptide expressed in vitro activated m TOR signaling pathway and Wnt / β-Catenin upstream signaling pathway.CCK-8 cell proliferation test revealed that the antibacterial peptides Reg3 a and Reg1 promoted the proliferation of IPEC-J2 and IEC-6 cells;two kinds of intestinal epithelial cell scratch test and Western blot showed that the antibacterial peptides Reg3 a and Reg1 promoted the migration of intestinal epithelial cells,and enhanced the expression of tight junction related proteins.In conclusion,recombinant antimicrobial peptides Reg3 a and Reg1 expressed in vitro can inhibit the proliferation of intestinal pathogens and enteroviruses,and promote the proliferation and migration of intestinal epithelial cells and the formation of tight junctions through mTOR signaling pathway and Wnt / β-catenin signaling pathway...