Structural Transformation Of Shigella Dysenteriae Serotype 1 O-polysaccharide And Study On Codonopsis Pilosula Endophyte Exopolysaccharides

Carbohydrate(including monosaccharides,oligosaccharides and polysaccharides)and glycoconjugate widely exist in various organisms.As important structural components and information molecular,carbohydrate plays a crucial role in many biological processes.Bacterial surfaces are coated with complex polysaccharides,including lipopolysaccharides(LPSs),exopolysaccharides(EPSs)and capsular polysaccharides(CPSs),among which LPS is covalently linked to cell surfaces and a particular composition of gram-negative bacteria in the outer membrane.It typically consists of lipid A,core oligosaccharide,and a distal polysaccharide(O-antigen).It is composed of many repeating units,has diverse structures,and is vital virulence factors of disease-causing bacteria.EPS is high molecular weight carbohydrate polymers secreted by microbial cells.O-polysaccharides(O-PS)belonging to the polysaccharide antigen is constituted by O-repeating units(up to 100 copies).O-PS covalently linked to core oligosaccharide and lipid A to form complete LPS.O-PS exhibits vast structural complexities.Based on structural complexities,bacteria are divided into different O-serotypes.Increasing evidences show that O-PS plays essential roles in bacteria-host interactions,and involved in bacterial infection.S.dysenteriae 1 causes severe shigellosis that is typically associated with high mortality.Antibodies against Shigella serotype-specific O-polysaccharide have been shown to be host protective.It has been reported that anti-carbohydrate antibodies are naturally present in large amounts in human serum,such as anti-Rha,anti-Man,anti-GalNAc.Researchers have focused on the anti-a-Gal antibody.For example,a-Gal epitope has been conjugated with influenza protein antigens,HIV gp120 and cancer antigens to boost their efficacy as vaccines.However,in recent years,more and more attention has been paid to anti-Rha antibody,because of the even more abundance of anti-Rha antibody in human serum than anti-a-Gal antibody.In addition,Rha is the general antigen on many pathogenic bacteria surface.Thus,the application of Rha for vaccine design is growing.According to the principle of affinity chromatography,we purified human anti-Rha antibody,which has strong specificity to Rha.In the second part of this thesis,the heterologous biosynthesis of S.dysenteriae 1 O-antigens in a bacterial carrier was performed.The glycosyltransferases responsible for the biosynthesis of O-antigen in S.dysenteriae 1,including rfbR(rhamnosyltransferase),rfbQ(rhamnosyltransferase)and orf9(galactosyltransferase),as well as rfp(galactosyltransferase)were cloned into pACT3 and pET15b,respectively.Then,the recombinant vectors were transformed into E.coli K-12 W3110 as a live,bacterial vaccine delivery system for heterologous O-antigens.Finally,LPS was isolated by hot phenol-water extraction procedure and purified by gel permeation chromatography on a column of Bio-Gel P-2 with water as eluent for structural analysis.LPS was examined by silver staining,western blot using human anti-Rha and S.dysenteriae 1 LPS antisera,matrix-assisted laser desorption-ionization and ion chromatography(IC).The heterologous expression of O-antigen containing Rha was confirmed by our results.In the third part of this thesis,the preliminarily study on Codonopsis Pilosula endophytes 14-DS-1 was carried out.Codonopsis Pilosula polysaccharide provides many heslth benefits,such as anti-age,anti-fatigue,anti-tumor and anti-radiation.The strain 14-DS-1 was isolated from Codonopsis Pilosula.Based on the amplified sequence of the nearly complete 16S rDNA gene of strain 14-DS-1,the phylogenetic affiliation of this strain was analyzed.The strain 14-DS-1 was classified as a sub-branch of the genus Bacillus.To study its EPS,the EPS was purified by Sephadex G-50 after precipitation from the fermentation broth with absolute ethanol and deproteination using the Sevage method.Monosaccharide composition analysis of the EPS was performed by IC.The results showed that it is composed mainly of fucose,glucose and minor peaks for the other four modified monosaccharide.Secondly,the cell activities of EPS produced by strain 14-DS-1 was preliminarily investigated.