Acinetobacter Baumannii Polysaccharide Conjugate Vaccine Research

Acinetobacter baumannii is a Gram-negative bacteria,opportunistic pathogen that widely present in nature and typically infects immunocompromised patients.It is also one of the most representative clinical pathogens in hospital infection.Because of its high proliferation rate and strong adhesion,A.baumannii widely distributed in the hospital environment.Besides with the extensive use of antibiotics,there have been more and more multiple or extreme drug resistance strains.A.baumannii has posed a serious threat to the global health system.At present,the clinical against A.baumannii infections major use of antibiotics.However,with the emergence of multiple drug-resistant strains,the doctor had to use in drug combination for the ill patients.There are many drawbacks of joint medication yet,so it is particularly urgent to the development of related vaccines for the prevention A.baumannii epidemic.Unfortunately,there is no one vaccine available for A.baumannii.Extracellular polysaccharides of Bacteria are an important part of the outer membrane structure,the O-polysaccharide(O-PS)has a specific immunogenicity,it is able to stimulate the protective antibodies production.But the individual polysaccharides belong to the T-cell-independent antigen,which produced antibodies mainly is low affinity of IgM.Furthermore,individual polysaccharides can not induce humoral immunity.And getting through combining the polysaccharide conjugate formed on a polysaccharide with a protein as a vaccine to induce a better immune response.The polysaccharide conjugate binds to B-cells,which is degraded and formed glycopeptides,it can be presented to the major histocompatibility complex MHC-II.The polysaccharide is shown to T-cells,activates the production of typical T-cell-dependent antibodies,inducing the production of polysaccharide-specific IgQ and Memory cells will proliferate.Therefore,this study utilizes the advantage of glycoprotein conjugates as immunogens,based on the synthesis of gene cluster of Acinetobacter baumannii itself,transfer the synthetic polysaccharide to the target substrate protein,one-step biosynthetic glycoprotein vaccine in pathogens.First,the protein glycosylation vector pET28a-PglL-CTB4573C,which has been constructed in the laboratory,was transferred into Acinetobacter baumannii 17978,construction of glycosylated engineering Acinetobacter baumannii successfully.Glycosylation of Acin.etobacter baumannii can express glycosyltransferase PglL and substrate protein CTB.PglL is a glycosyltransferase of Neisseria meningitides,which is lower requirements on sugar chains and substrate proteins.CTB is a cholera toxin(CT)B subunit that is capable of forming homologous pentamers.While CTB as a carrier protein,the fusion epitope antigen will greatly enhance the immunogenicity,stimulate the body to produce a strong immune response.After the successful construction of glycosylated engineering Acinetobacter baumannii,through the induction of expression,obtained with Acinetobacter baumannii 17978 specific polysaccharide as the target antigen.CTB as the substrate protein specific glycoprotein complex,utilizes the nature of the substrate protein itself and carried the His tag,We can carried out to obtain the pure target protein by affinity chromatography and cation exchange chromatography,and then the purified glycoprotein was evaluated for immunogenicity.Select mice as model animals,according to the results of sugar quantitative,2.5μg for one mice immunization.This study used both abdominal and subcutaneous immunization,the titer of serum antibody was measured by indirect ELISA.Both the peritoneal and subcutaneous groups,the glycoprotein can stimulate mice to produce IgG,IgG1,IgG2a,IgG2b,IgG3 antibodies.Indicating that glycoprotein can induce the body to produce cellular immune response and humoral immune response.Finally,the mice were challenged intraperitoneally to experimental attack toxic dose of 2 × LD50 observed continuously for 7 days after challenge.The results showed that the protective effect of the peritoneal group was 100%,but the subcutaneous group had no significant protective effect.We found that the glycoprotein expression quantity was lower expression at protein purification,which brings difficulties for production and post-experimental drugs.Therefore,according to the synthesis pathway of extracellular polysaccharide of Acinetobacter baumannii,the glycosyltransferase PglLAb gene of Acinetobacter baumannii itself was knocked out by homologous dual exchange method,in order to reduce its own glycosyl transferase and exogenous PglL competitive,so as to improve the output of glycoprotein.This study constructed the glycosylated engineering Acinetobacter baumannii,and its production of glycoprotein purification,through animal experiments proved that glycosyl-binding vaccines produced by Acetobacter baumannii have a good immunogenicity.It brings to the follow-up research necessity.On this basis,the tapping of Acinetobacter baumannii glycosyl transferase PglLAb gene,in order to the application of the next and vaccine production laid the foundation.