Preparation Of Monoclonal Antibody Against Suilysin Of Streptococuccs Suis Type 2 And Identification Of It's Epitopes

Streptococcus suis(S.suis) is a common pathogenic microorganism and can cause a variety of clinical diseases in human and swine.Streptococcus suis mainly causes meningitis and occasionally causes other infections,such as endocarditis,arthritis,and pneumonia.Based on the capsular polysaccharide antigens of S.suis,35 serotypes or capsular types have been characterized.S.suis serotype 2(SS2) is most commonly associated with severe disease and is the serotype most frequently isolated from pigs.Str.Suis type 2 produce such virulence facters as suilysin(SLY),muraminidase protein(MRP), extracellular facter(EF),ect.The haemolysin was produced at the end of the exponential growth phase.It belongs to the family of toxins known as antigenically related cholesterol-binding cytolytic toxins,since it shares common characteristics with other members of this family,such as sensitivity to oxygen and oxidizing agents,activation by reducing agents,inhibition by low concentrations of cholesterol,formation of transmembrane pores and a multihit mechanism of action.In addition,anti-streptolysin antibodies inhibited the haemolytic activity caused by the S.suis haemolysin.The pathogenesis of S.suis infection has not been completely defined.However,in order to cause meningitis,S.suis has to cross the blood-brain barrier(BBB) made up of brain microvascular endothelial cells.At high bacterial doses,suilysin-positive strains were toxic for porcine brain microvascular endothelial cells.The role of suilysin in cytotoxicity was confirmed by using purified suilysin,electron microscopy,and the lack of toxicity of a suilysin-negative mutant.In swine,the invasion of endothelial cells of the BBB could play an important role in the pathogenesis of the meningitis caused by S.suis.In order to study SLY biologic function,the following research were explored.1.Cloning of SS2 SLY gene and it's expression in E.coli preparation of Monoclonal Antibodies against SS2 SLY.SLY gene was amplified by PCR technique from Streptococcus type 2 strain,SLY was inserted into downstream of the T7 promoter of an expression vector,pET-28a,to yield the recombinant plasmids pET-28a-SLY,and pET-28a-SLY was sequencued by Sanger's sequencing technique.Then it was expressed in E.coli BL21(DE3)after induced by IPTG.A high expression level of fusion proteins was obtained.SDS-PAGE analysis showed that the fusion proteins was 57KD in size.SLY existed mainly in form of inclusion bodies and was specific to antisera against SLY by western blot analysis.Two hybridoma clones were generated by fusion of SP2/0 myeloma cells and the splenocytes from BALB/C mice-immunized with rSLY.The 2 hybridoma clones(1A11,4D1) were obtained.The ELISA titers of the ascite fluid induced by these 2 hybridomas were 100×2⁹ and 100×2¹⁰ respectively.The results of indirect ELISA and Western bloting indicated that these two monoclonal antibodies could react specifically with the corresponding Suilysin of Streptococcus suis 2.Further characterization revealed that both 1A11 and 4D1 strains belong to IgG2a isotype,and the light chains were k chains.The two strains of monoclonal antibodies could restrain the solvation of erythrocyte.2.Screening of a mimic peptide for SLY from phage displayed 12-mer peptide libraryPhage display is a advanced molecular technology that allows the presentation of large peptides or protein on the surface of filamentous phage by fusing sequence of a target gene with the phage capsid gene.A crucial advantage of this technology is relatively independent three dimensional structure and biological activity of the displayed peptide or protein,which allows directly selection and study of functional domains of binding parteners such as antigen-antibody or receptor-ligand.Monoclonal antibody 1A11 was used to screen for binding peptides form a phage display 12-mer peptide library.After three rounds of biopanning,the majority of the selected clones were found able to react to McAb 1A11,but not to BSA.The amino acid sequences of clones binding to McAb 1A11 contained an identical sequences W-P-WD, sequence comparisons suggest that it may have evident homology to 453-459 amino acid between the SLY.3.Investigating of SLY epitopes deletion mutantIn order to investigate the role played by SLY epitopes in SLY biologic function,we generated a defined deletion mutant of the SLY in epitopes and compared the wild-type SLY protein with the SLY in mutant in a number of assays.Compared with wildtype SLY,mutant SLY with deletion of several amino acid was not hemolytic.The Western blot assays of deletion epitopes proteins raised against SLY antibody were positive,while for the assay against SLY McAb were negative.