Effect Of AI-2/LuxS Quorum Sensing System On S-layer Protein Expression Of L.acidophilusCICC6074

L.acidophilus is a kind of probiotics that can colonize the human intestinal tract.The ability of its colonization depends mainly on the adherence of its S-layer protein to intestinal epithelial cells.Quorum sensing system(AI-2/LuxS)is a way of communication between bacteria through signaling molecules,widely existing in a variety of gram negative bacteria and gram positive bacteria.A large number of studies show that LuxS mediated quorum sensing system plays an important role in the formation of biofilm,but the study of L.acidophilusCICC6074 mediated AI-2/LuxS quorum sensing system has not yet been reported.The effect of AI-2/LuxS quorum sensing system on the expression S-protein in L.acidophilus CICC6074 was studied in this paper.The main methods and results of our research are as follows:1.Molecular cloning technique was used to test whether L.acidophilusCICC6074 existed luxS gene and whether it could produce quorum sensing signal molecule by molecular biology method AI-2.Gene was sequenced and compared to L.acidophilusNCFM.Using multifunctional enzyme standard mode of chemiluminescence detected whether L.acidophilusCICC6014 produces signal molecules.Comparison showed that the similarity of L.acidophilusCICC6074 and L.acidophilusNCFM is 100%;L.acidophilusCICC6074 can produce AI-2 signal molecules with activity,with the growth of cell density the AI-2 signal molecular also increased,reached the maximum value in the middle of the log.The first preliminary research proved that L.acidophilusCICC6074 gene can catalyze AI-2 signal molecule synthesis and produce AI-2 signal molecules with vitality.2.The slpA gene fragment was amplified by PCR and transformed it into E.coli.Then cells was broke to obtain SlpA protein.Protein purification kit of the protein was purified and injected into rabbits in vivo immune response.The rabbit-derived antiserum was collected after the 4th immunization and purified by antigen coupling sepharose chromatography.Rabbit serum before immunization was collected for control.The titer of polyclonal antibody was assessed by indirect enzyme-linked immunosorbent assay(ELISA).The results showed that the blank and negative control OD<0.2,purified by affinity chromatography after OD1:64K>1,ELISA titer>1:64K.the results meet the requirements.3.This study designed specific primers to amplify luxS genes and linked luxS genes to pHL460 expression vector.Then transformed it into L.acidophilusCICC6074 and extracted induced proteins to do qualitative detection.The expression of luxS gene was detected by qPCR,and the expression level of LuxS protein and S-layer protein was detected by WB.LuxS gene expression vector pUS460 was successfully constructed,and the gene could be expressed in L.acidophilusClCC6074.The increase of LuxS protein expression caused expression of S-layer protein in L.acidophilusCICC6074.It is proved that AI-2/LuxS quorum sensing system can mediate the expression of S-layer protein in L.acidophilusCICC6074.In summary,the AI-2 was detected by L.acidophilusCICC6074.The effect of luxS gene on the expression of S-layer protein was detected by qPCR and Westernblot,which laid the foundation for further research on the probiotic properties of L.acidophilusCICC6074 mediated by AI-2/LuxS.