| Haemophilus parasuis (H.parasuis) is the pathogen of Gl sser’s disease, the main symptomsinclude pericarditis, polyarthritis, multiple fibrinous serositis and meningitis, it can also cause sepsisorwithout polyserositis pneumonia. It can be isolated from the nasal cavities of healthy pigs. Gl sser’sdisease can result in high morbidity and mortality in non-immune pigs such as the nursery pigs.H.parasuis has become an important pathogen in the pig industry around the world, the infectionscaused by the bacteria is very common, and result in higher economic losses to the pig industry.Immunohistochemistry (IHC), oligonucleotide-specific capture the the plate hybridization test(OSCPH), polymerase chain reaction (PCR) have been used in detecting pathogen of H.parasuis.Complement fixation test (CFT), enzyme-linked immunosorbent assay (ELISA), indirecthemagglutination (IHA) have also been applied to serological detection of H.parasuis. Polyclonalantibodies (PcAb) prepared by the whole bacteria is difficult to avoid the interference of proteinconserved between species, so there is still some phenomenon of cross-reactivity. In addition, themolecular diagnostic techniques based on the16S rDNA is also difficult to avoid the interference ofActinobacillus indolicus (A. indolicus). In short, lack of understanding to the specific antigen proteinand nucleotide sequence about H.parasuis blocked the practical applications of the diagnostic methoddescribed above. Therefore, the identification of specific antigen among interspecies which isconservative intraspecific is particularly important. These specific antigens will promote the applicationof the diagnostic methods in the diagnosis of H.parasuis.In this study, we prepared monoclonal antibody (MAb) with high specificity and stability.Identification of the specific proteins and epitopes recognized by the MAb laid a good foundation forthe diagnosis of H.parasuis.To screen valuable MAb for the clinical diagnostic of H.parasuis infection, virulent strain HPS-5was used to immunize BALB/c mice. A stable MAb was successfμLly generated, Western blot analysistestified that MAb1B3specifically identified all standard strains of H.parasuis but not Salmonellaenterica (S.enterica), Staphylococcus aureus (S. aureus), enterotoxigenic E. coli (ETEC), lactic acidbacteria (LAB) and serotype1Actinobacillus pleuropneumoniae(APP-1). Immunoprecipitation andprotein spectrum showed that MAb1B3identified the oligopeptide-binding protein A (OppA) whichbelongs to the ATP binding cassette transporter (ABCT) family. Amino acid sequence analysis of OppAshowed that H.parasuis and other ten severe swine bacterial pathogens have a identity percent from9.1%to74.5%. This provides a good basis for OppA to be used as a potential diagnostic marker.In order to further identify specificity epitope in OppA, we did three panning of the phage12-merpeptide library through the MAb1B3, fifteen positive phage clones were obtained. Sequence analysisshowed that eight phage clones displayed a conserved sequence KTPSE-R corresponding to469KTPAE-R475of H.parasuis OppA.To verify the results above and the reactogenicity of OppA, we expressed a fusion protein andsynthetized a series of15amino acids peptides respectively which contain conserved sequences in phage display results. Western blot analysis of a truncated fusion protein and ELISA analysis of the15amino acid sequence verified the result described above, and we also found epitope of OppA which is asfew as6amino acids.In conclusion, this study has a certain reference value to optimize diagnostic methods ofH.parasuis and to the development of differential diagnosis reagent. |
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