Cloning And Expression Of Glutamate Dehydrogenase Of Streptococcus Suis Serotype 2, A Preliminary Study Of The Monoclonal Antibody And Nucleic Acid Vaccines

Streptococcus suis is an important pathogen which has been associated with a wide variety of infections in swine, such as meningitis, septicemia, arthritis, and pneumonia . This organism has also been isolated from humans with meningitis or endocarditis. Several putative virulence factors have been described,including the capsular polysaccharide, muramidase-released cell-wall protein, extracellular protein factor, suilysin. Virulence factors of S. suis type 2 are not well characterized. One of the factors thought to be involved in the pathogenicity of this bacterium is the production of proteases, which are,in turn, regulated by the levels of nitrogen available.Glutamate dehydrogenase (GDH) is a key enzyme that links carbohydrate (energy) and nitrogen metabolism .1 Characterization,cloning,and in vitro expression of the glutamate dehydrogenase of Streptococcus suis serotype 2We amplified gene of GDH from Habb chromosome by PCR and then cloned it into prokaryotic express plasmid pGEX4T-2,and the recombinant plasmid pGEX4T-2-gdh was transformed into E.coli BL21(DE3). After the induced expression, the protein GDH was isolated and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Then the fusion protein was primary purified by affinity chromatography. The GST tail of the fusion protein was cleaved by Thrombin. Nucleotide sequence analysis showed that the gdh gene of S.suis possesses the highly conserved motifs typical of family I hexameric GDHs. The results of SDS-PAGE showed that the molecular weight of the GST fusion protein was 71kDa, and the one of cleaved protein GDH was 45kDa. 2.Identification the activity and immunogenic of the recombinant protein GDHGDH activity was assayed spectrophotometrically by following the decrease in A340 during oxidation of NADPH or NADH.We found that the GDH of S.suis belongs to Enzyme Family I, and its activity is attributed to NADP-dependent GDH (GdhA). To examine the extent of conservation of the gene among the S. suis capsular types, PCR was used to amplify the DNA of the native S. suis strains encompassing all serotypes .The primers amplified the DNA from strains of most serotypes and produced a fragment of the expected size,except serotypes 22,26,27,29,32 and 34. In order for the recombinant protein to serve as a candidate for the development of a diagnostic reagent, the antibody produced against the protein must be reactive with a similar protein from a wild-type S. suis strain(s),means it must be immunogenic.We used western blot to prove it. 3 Production of monoclonal antibody against GDHFour monoclonal antibody cells named 4D6,1D10,2B9 and 3E10 were produced by fusing SP2/0 cells and spleen cells of BALB/c mice that were immunized with expression protein GDH and S. suis strain ZY05719. The positive wells were screened by enzyme-linked immunosorbent assay with purified GDH as antigen.Cells were monoclonalized by limiting dilution.All four monoclonal antibodies were...