The Adhesion And Clonization Mechanism Of Lactobacillus Isolated From The Intestinal Tract Of Paralichthys Olivaceus

Lactobacillus as a kind of probiotic,plays an important role in healthy culture and disease prevention of aquaculture.However,most clinical studies of probiotics persistence and colonization show that they do not permanently colonize the gastrointestinal tract and continue providing their benefits to hosts only for brief periods after stopping feeding.In order to study the adhesion and colonization mechanism of Lactobacillus in intestine,two strains of Lactobacillus were isolated from the intestines of healthy wild flounder,and their adhesion-related proteins were identified by biotinlabeling,Western blotting and high performance liquid chromatography mass spectrometry(LC-MS/MS).It provides data support and theoretical basis for exploring the colonization and adhesion mechanism of Lactobacillus in the intestinal tract of P.olivaceus.The dissertation included the following five parts:(1)Isolation and identification of Lactobacillus,including Gram staining,physiological and biochemical identification,16 S r DNA sequence alignment,acid resistance,bile salt resistance,surface hydrophobicity determination.The results showed that the two strains of L.plantarum(LPPO23)and L.sakei(LS-PO11).LS-PO11 has the higher acid resistance,bile salt resistance and hydrophobicity.(2)The immunomodulatory ability of Lactobacillus to the host.The results showed that both LS-PO11 and LP-PO23 could increase the expression of five immune-related genes in Gill and head kidney after feeding Lactobacillus.The results showed that both LS-PO11 and LP-PO23 could significantly increase the expression of immune-related genes IL-1β,TNF-α,MHC-II,Ig M and TCR-β in Gill and head kidney after feeding with Lactobacillus.In Gill,the expression of IL-1β,TNF-α,Ig M and TCR-βgenes reached the peak earlier after feeding LP-PO23 than that of LS-PO11.In the head kidney,after feeding LP-PO23,the expression of TNF-α,MHCII and Ig M genes reached the peak ear lier than that of LS-PO11.(3)Adhesion of Lactobacillus to epithelial cells ability.The results showed that the adhesion of LP-PO23 to intestinal epithelial cells was higher than that of LS-PO11,and the adhesion of two Lactobacillus strains decreased si gnificantly after Li Cl treatment,indicating that there were proteins related to adhesion and colonization on the surface-layer.Realtime quantitative PCR detection showed that during the feeding of Lactobacillus,the number of LS-PO11 、 LP-PO23 colonization in the intestinal tract of P.olivaceus increased significantly,and the colonization level of LP-PO23 was higher than that of LS-PO11.After stopping feeding,the retention time of LP-PO23 in the intestine was longer than that of LS-PO11.(4)The difference of surface protein(Surface-layer proteins,SLPs)of Lactobacillus.The surface proteins of Lactobacillus were extracted by 5 M Li Cl and 2M guanidine hydrochloride respectively and analyzed by SDS-PAGE.When used the same extraction method,the surface protein bands of LS-PO11 and LPPO23 are different,there are specif ic bands,indicating that there are differences in the surface protein components of the two strains.Compared with 5 M Li Cl,the abundance of protein extracted by 2 M guanidine hydrochloride was higher,and some bands only existed in the surface protein extracted by guanidine hydrochloride.(5)Using biotin labeled membrane protein of intestinal epithelial cells of P.olivaceus.Lactobacillus adhesin was identified by Western blot combined with LCMS/MS.The results showed that among the 12 specific bands,LP-PO23 adhesins include chaperone Dna K 、 chaperone 60 k Da 、 enolase,elongation factor Tu 、phosphoglyceraldehyde dehydrogenase and lactate dehydrogenase.LS-PO11 adhesins include chaperone CLPB、transpeptidase、peptidoglycan hydrolase、chaperone 60 k Da、enolase and elongation factor Tu.