Characteristics And Gut Immune Function Of Lactobacillus Acidophilus S-layer Protein

Lactobacillus is normal microflora in the digestive tract and genital tract of human and animals,they can improve the ecological balance of flora and improve immune function.S-layer protein is an important protein molecule on its surface.At recent years,several studies have found that the S-layer protein has biological antagonism against the enteric pathogenic bacteria.By adhesion,invasion,leading to cytoskeletal rearrangements and affecting the signal transduction pathway,enteric pathogens can induce epithelial cell apoptosis so as to achieve the purpose of the invasion and have an bad effect on body function of animals and human.Therefore,inhibiting and effectively preventing the adhesion,invasion of pathogens to the host cell is the main way to protect cells from infection.Lactobacillus S-layer protein is the smallest S-layer protein known before,the molecular weight is between 25?71 kDa and it is alkaline(pI>9).Lactobacillus S-layer protein's structure is now in the exploratory stage,and most of its functions are unclear.On the basis of the extraction and identification of Lactobacillus acidophilus S-layer protein,this study use Caco-2 cell model to explore the inhibition effect of Lactobacillus acidophilus and its S-layer protein on pathogen bacteria adhesion.The effect of Lactobacillus acidophilus and its S-layer protein in the intestinal mucosa function on pathogenic Escherichia coli(EPEC 086:K61)infected mice was also investigated.It aims at confirming the adhesion properties of S-layer protein and providing a theoretical basis for the following studies of its adhesion mechanisms.The main contents and conclusions in this dissertation were summarized as follows:1.Lactobacillus acidophilus CICC 6074 S-layer protein was extracted by 5mol/L lithium chloride(5M LiCI)(pH=2)and separated and purified by using gel filtration chromatography Sephadex G-75;furthermore,the S-layer protein obtained with biological activity is electrophoretic purity.Combined with mass spectrometry we concluded that the molecular weight of S-layer protein is 43 kDa and its isoelectric point(pI)is 9.43.The heat distortion temperature and secondary structure of S-layer protein were also evaluated.CD experiments showed,a-helix and ?-folding were 16.9%and 42.3%in S-layer protein secondary structure,and about 40%of P-turn and random coil;DSC results showed that S-layer protein has two Tm respectively around 38?and 92?.2.Based on the single-factor test,the comparing experiment proved that the S-layer protein played an important role in the adhesion of Lactobacillus acidophilus on Caco-2 cells and found the S-layer protein can bind the cell wall reversibility.It is showed that S-layer protein can inhibit the invasion of pathogenic E.coli and the inhibition is dose-dependent in a certain range of concentrations;it can significantly reduce the invasion of EPEC(P<0.01)at a concentration of 100?g/mL and 150?g/mL.The exclusion,competition and displacement experiment of adhesion and invasion inhibition test of Lactobacillus acidophilus and S-layer protein all showed that Lactobacillus acidophilus have a better adhesion and invasion inhibition effect on EPEC than Lactobacillus acidophilus without S-layer protein,while exclusion test has a better inhibition rate.Lactate dehydrogenase(LDH)and cell proliferation activity tests showed that S-layer protein can help to reduce LDH activity of Caco-2 cells culture supernatant and to maintain cell viability infected with EPEC.Compared with none S-layer protein Lactobacillus acidophilus treatment groups,the inhibition of Lactobacillus acidophilus treatment groups is more significant:LDH activity in each time period reduced by an average of about 12.62%(p<0.05);cell activity relatively increased 5.12%.3.Through animal experiments we found that S-layer protein helps to inhibit the pathogen invasion,to reduce inflammation reaction and to protect intestinal mucosal immune function:The content of mucosal SIgA and serum IL-10 increased obviously after intragastric administration of Lactobacillus acidophilus compared with infection groups,they respectively increased about 20.22%and 11.25%compared to none S-layer protein Lactobacillus acidophilus gavage groups;And the content of serum TNF-??IFN-? and IL-1? decreased significantly compared with infection groups,they respectively decreased by approximately 8.31%,11.42%and 10.75%compared to none S-layer protein Lactobacillus acidophilus gavage groups.