Effects Of Gene-knock Out On The Physiology Of The Bacteria And Synthesis Of Glycoprotein By One Step

E.coli O157:H7is one kind of the enterohemorrhage Escherichia coli (EHEC), which can spread diseases mainly via food and water and causes diarrhea, hemorrhagicenteritis, hemolytic uremic syndrome and so on. As the primary structure on the surface of bacteria, LPS plays parts in recognition and infection of the host and is the major pathogenesis factor of gram-negative bacteria. O-polysaccharide antigen is the important component of LPS. It could break through the immune system of host and induce the specific immunoreaction. The specificity of O-polysaccharide determines the specificity of immune response.In the E.coli Wzy-dependent pathway, oligosaccharide chains are linked to the core oligosaccharide of lipoid A, which is catalyzed by O-antigen ligase WaaL, to form LPS. Saccharide chains are the significant components of flagella and pili on the surface of bacteria. Flagella enhance the adherence of bacteria to the end of the epithelial cells of the rectum of host not only because of its motility or chemotaxis, but also flagellum itself is the important adhesion organ。Bacteria processes duplication and infection via motility, swarming, twitching and so on in its hosts.We knock out the Wall gene in E.coli O157:H7to study the influences of LPS deficiency on flagellum itself, the physical condition, motility and adherence of bacteria, and the change of biological membranes. By expressing Wall in E.coli O157:H7△wraaL, we attempt to clarify the impacts of LPS on physical condition of bacteria. Our results provide supports to the development of medical reagents, which target the LPS via the LPS deficiency and recovery experiments.Saccharide which is one of the three biggest organics, has essential functions in the metabolic process and meanwhile participates in structure composition of living benings.. Saccharide involes life action by various ways,such as oligosaccharide, polysaccharide.glycoprotein and glycolipid. It is necessary to research saccharides for disease treatment and drug development because occurrence of some disease is closely correlated with the changes of saccharides,structure or number.As the important component of glycoconjugate, glycoprotein has two forms: N-glycosylation glycoprotein and O-glycosylation glycoprotein. Previously,people thought that N-glycosylation only exists in eukaryotes. In1990, Szymanski et al found a pgl gene cluster encoding N-glycosylation modification pathway in Campylobacter jejuni and then confirmed that N-glycosylation also exists in prokaryotes. pglB, responsible for transferring oligosaccharide chain to receptor protein AcrA, is the key enzyme of pgl gene cluster and has low substrate specificity. Although there are similities in oligosaccharide chains synthesis and carbohydrate chains flip between N-glycosylation in C.jejuni and O-PS synthesis of Wzy-dependent pathway in E. coli,but in C.jejuni the oligosaccharide is transferred to AcrA while in the Wzy-dependent pathway of E. coli it is linked to the core oligosaccharide of lipoid A to form lipopolysaccharide (LPS). The high similities of oligosaccharide chains synthesis and carbohydrate chains flip provise possibilities for us to replace oligosaccharide chain of C.jejuni in itself with O-PS.In the synthesizing process of Wzy-dependent pathway in E. coli, O-antigen ligase WaaL links oligosaccharide chains to the core oligosaccharide of lipoid A. By gene knockout technique, we knock out the waal gene in E. coli O86:K61:B7,making the O-PS which is due to linked to the core oligosaccharide of lipoid A is accumulated in periplasmic space. Then by gene recombination,we express pglB in E. coli O86:K61:B7of which waal gene has been knocked out, and futher the N-glycosylation pathway expressed in E. coli could make O-PS of bacteria and receptor protein form glycoprotein. We could select different proteins as substrate because of the loose substrate specificity of Pg1B. Maltose-binding protein (MBP),which is prevalent in periplasmic space, could transfer maltose or maltose dextrin to MalFGK2.As a tag, MBP also can improve the solubility and stability of the target protein. There is "permissive site"in the location of178and341residues of MBP,and insertion or mutation of70amino acids in these two locus would not affert the activity and conformation of MBP. When as the receptor protein of vaccine,MBP has the ability to bind Toll-like receptor(TLR4) and promote the activation of Thl-type cytokine to activate antigen presenting cell-dendritic cell(DC), and ultimately stimiulate the specific immune response and immunological memoryof human. In view of these advantages above, we select MBP as the receptor of N-glycosylation patway catalyzed by pglB.Conservative senquence(for example WWD motif) and spatial conformation of pgIB determine that the best N-glycosylation modification loci is DQNAT pentapeptide. By virtue of gene-mutation and fusion PCR techniques, we obtain the DQNAT N-glycosylation modification locus in the178and341residues of MBP. The178and341amino acid residues lie at the flexible and easy-folding nonregular coils, which are on the surface of MBP crystal structure. This provides feasibility for pglB to make use of MBP as substrate to catalyze glycosylation reaction.In this paper, we use Pg1B to construct N-glycosylation system in E.coli O86:K61:B7and remould non-natural substrate protein MBP to make it become the substrate of glycosylation for the first time. Now, we can synthesize glycoprotein conjugate by one step in biological systems, overcoming the problems, such as contamination, waste and complex, in the production of glycoprotein conjugate by chemical method.