Study On Preparation And Immunological Properties Of Hcp1 And Polysaccharides From Burkholderia Pseudomallei

Burkholderia pseudomallei,a facultative intracellular gram-negative bacterium mainly exists in water and soil,has been listed as a category B bioterrorism agent by the WHO.Humans and animals acquired infections through a variety of ways including respiratory tract,digestive tract,etc.,and causing a kind of melioidosis of zoonotic disease.The clinical symptoms of melioidosis are diverse,including refractory pneumonia,gangrene,and fatal sepsis.B.pseudomallei was proved to be naturally resistant to widely-available antibiotics,therefore the treatment is often prolonged,and the recrudescence is common.At present,there is no vaccines are available to protect against melioidosis,thus,it is also challenged to prevent and treat of this disease.The results of the survey show that the diagnosis of melioidosis is seriously underreported in the world.At present,laboratory diagnostic methods（such as bacteriological culture,molecular biology methods and proteomics methods）for B.pseudomallei are limited,and there is no commercial and reliable rapid diagnostic method for B.pseudomallei in clinical applications.Therefore,it is urgent to establish a simple and rapid diagnosis method.Enzyme-linked immunosorbent assay（ELISA）,a standard serological testing method,is the basis for further development of rapid testing methods.Previous studies have shown that the expression level of B.pseudomallei hemolysin coregulated protein 1（Hcp1）increase significantly during the process of infection of the host.The evidence indicated that Hcp1have a strong antigen-antibody reaction with the serum of patients with melioidosis,which suggests that Hcp1 can induce the body to produce specific antibodies and could be used as a serological diagnostic candidate antigen.From what has been discussed above,DNA recombination technology was used in this study,and a high-purity r Hcp1 protein with a molecular weight of about 18 k Da was obtaied through prokaryotic expression.Furthermore,the immunological properties of r Hcp1 protein were studied.Firstly,the indirect ELISA test was applied to detect the titer of antiserum.The results showed that r Hcp1 protein has high immunogenicity,with a titer of up to 1:512 000.Then the specificity of r Hcp1 protein and its antiserum was studied by western blotting and immunofluorescence experiments.The findings of this study suggested that r Hcp1 protein can never bind to the serum from healthy donors but only specifically bind to the serum of patients with melioidosis.Similarly,anti-r Hcp1 serum only specifically bound to different clinical B.pseudomallei strains,but does not react with other pathogens.Therefore,we deduced that r Hcp1 protein can be used as a candidate antigen target for the serological diagnosis of melioidosis.Lipopolysaccharides（LPS）,capsular polysaccharides（CPS）and exopolysaccharides（EPS）are the main surface-associated antigens of B.pseudomallei,are essential to bacterial virulence.These polysaccharides play an important role in the pathogenic mechanism and immune regulation,and have the potential as diagnostic antigens and vaccine candidate antigens.Since the bacteria can express a variety of LPS and CPS,so far,there are no relevant reports on the surface polysaccharides of clinical isolates of B.pseudomallei in our country,thus,the types of polysaccharide antigens and their biological functions are still unclear.Based on this,a series of studies were carried out in this study,including purification,structural characterization and immunogenicity of outer membrane polysaccharides of clinical strains derived from different clinical specimens.First,the extracellular polysaccharides of different clinical strains were obtained by a stepwise extraction method,and then were separated and purified by ion exchange chromatography and gel-permeation chromatography chromatography to give three purified fractions BPC004-BPPI-b1,BPC006-BPPI-b1 and BPC010-BPPI-b1.High performance gel permeation chromatography（HPGPC）results showed that the three purified antigen components（BPC004-BPPI-b1,BPC006-BPPI-b1 and BPC010-BPPI-b1）were relatively uniform in composition,and their molecular weights were931 k Da,640 k Da and 546 k Da,respectively.Furthermore,the structure of these purified antigens were characterized by GC-MS,methylation analysis,FT-IR and 1D/2D NMR.The results indicated that the structure of BPC004-BPPI-b1 was composed of[3)-2OAc-β-D-6-deoxy-Hepp-（1→3）-β-D-6-deoxy-Hepp-（1→]repeating units,meahwhile,BPC006-BPPI-b1wasmainlycomposedof[3-（a-D-Manp-1→3-a-D-Manp）₄-2Me-a-L-6d Talp-1→].The structure of BPC010-BPPI-b1 is basically the same as BPC006-BPPI-b1.Furthermore,a preliminary study on the immunological properties of three polysaccharide antigens were carried out,besides,the relationship between the chemical properties of the epitope unit and immunological activity were further determined.Firstly,the immunogenicity and immunoreactivity of the polysaccharide antigens were studied by the indirect ELISA assay,and the results showed that three polysaccharide antigens have good immunogenicity,and specifically bound to the sera of patients with melioidosis,but not with the sera of healthy donors.Whereafter,the cross reactivities of antisera against three polysaccharide antigens with different clinical strains were determined by the indirect ELISA method.The results showed that each of the antiserum exhibited cross-reactiton with 80%of the clinical strains in this study,thus we deduced that[3）-2OAc-β-D-6-deoxy-Hepp-（1→3）-β-D-6-deoxy-Hepp-(1→]and[3-（a-D-Manp-1→3-a-D-Manp）₄-2Me-a-L-6d Talp-1→]may have great application value in the serological diagnosis of melioidosis and vaccine development.Finally,FT-IR,immunoblotting and competitive ELISA were used to study whether the acetylation modification group on the polysaccharide epitope could affect its immunoreactivity.The results showed that acetylation modification located on the epitopes seems to be important for their immunogenicity since the deacetylated antigens lose their ability to bind to the antibodies.In summary,the research results in this study will provide new theoretical basis and data support for the screening of melioidosis diagnostic antigens and vaccine candidate antigens.