Biosynthesis Of Glycoprotein By E. Coli And Exploration Of Regulating Polysaccharides Chain Length

Lipopolysaccharide (LPS) is 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).O-antigen belonging to the polysaccharide antigen is composed of multiple repeat units with the same structure. O-antigens can be divided into three groups according to the length of polysaccharide chain, namely long type O-antigen (19-25 repeat units), intermediate type O-antigen (10-18 repeat units) and short type O-antigen (<10 repeat units). Different species of bacteria comprise varying numbers of repeat units and the structures of O-antigen repeat unit can differ in the monomer glycoses, the position or stereochemistry of the O-glycosidic bond.LPS is able to stimulate the human immune system and cause a series of immune reactions. LPS, especially O-antigen part, is considered to be used in vaccine development based on its immunogenicity. The antigenicity of pathogenic bacteria is closely related with the length of O-antigen chains. O-antigen is locates in the surface of bacteria and can affect the function of some secretory proteins. It also influences the way how gram-negative bacteria interact with complement proteins. Moreover, the O-antigen of Gram-negative bacteria play an important role in the signal identification, adhesion, immune evasion and other processes. It is believed that long O-antigen chains provides a fitness advantage by conferring stronger resistance to complement.According to the type of the flippase that is involved, There are three kinds of biosynthesis mechanisms of O-antigen including Wzy-dependent pathway, synthase-dependent pathway and ABC-transporter dependent pathway. In Wzy-dependent mechanism, the wzz gene plays a key in regulating the length of polysaccharides resulting in various antigenicitys of the pathogen and immune responses of the host. This is important in developing vaccines with different antigenicity.It becomes possible that glycoprotein with O-antigen are produced in bacteria due to the similarities between the N-glycosylation pathway in Campylobacler jejuni and the Wzy-dependent O-antigen synthesis in Escherichia coli. In the second part of this thesis, the study is mainly about the biosynthesis of glycoprotein. The polysaccharide part is from the O-antigen of E. coli O157:H7. Firstly, we knocked out waal gene in the stain of E. coli W3110 based on the technique of Red homologous recombination.Secondly, we cloned E. coli O157:H7 O-antigen gene cluster in a vector named pYES1L-0157 rfb. Maltose-binding protein (MBP) is normally used as a novel carrier protein for glycoconjugate vaccines because of several advantages. For instance, MBP has signal sequence to periplasm and harbors TLR4 agonist-like properties which can induce the activation of NF-κB signaling pathway and secretion of proinflammatory cytokines. In addition, MBP could be easily expressed, glycosylated and proved. Therefore, we chose MBP as the carrier protein for our glycoconjugate vaccines. In order to enhance the efficiency of glycosylation, we inserted four glycosylated sites (DQNAT) at the C-terminal of the MBP sequence and cloned the gene of pglb and mbp in the same vector pBAD24. Finally, the strains that produce glycoproteins were constructed. The glycoproteins were verified by Western blot and MALDI-TOF-MS. By using bacterial protein N-linked glycosylation system, the synthesis of glycoproteins is simple and avoids suffering variable batch-to-batch composition, difficult quality control, inconsistent potency and high production cost in traditional chemical technologies.In the third part of this thesis, the study is mainly about the biosynthesis of glycoprotein with different antigenicitys. Firstly, we knocked out wzz gene in the stain of E. coli W3110Δwaal based on the technique of Red homologous recombination. Secondly, we cloned wzz genes from different strains containing various lengths of O-antigen polysaccharide to the pEXT22 vector respectively.Specifically, we chose the wzz genes from E. coli O111 and E. coli O127 that owned long O-antigen polysaccharide chains, and the wzz gene from E. coli O86:H2 that owned intermediate O-antigen polysaccharide chains. Thirdly, the technique of homologous recombination was used to clone E. coli O157:H7 O-antigen gene cluster (without wzz gene). Finally, the strains that produce glycoproteins with different lengths of polysaccharides were constructed and the glycoproteins were purified.