Determination Of The Functional Domain Of IMPDH Gene Of Streptococcus Suis Serotype 2

Streptococcus suis serotype 2 was the important pathogen of diseases in pigs and was amajor cause of meningitis, septicemia, arthritis and bronchopneumonia in young pigs andcould cause meningitis in humans.With Streptococcus suis serotype 2, pathogenic,weakly pathogenic,andnonpathogenic strains can be found.The virulent factors that can be found includemurarnidase-released protein(MRP), extracellular protein factor(EF),Suilysin(SLY),NewORF2, fibronection(Fn), glutamate dehydrogenase(GDH),secretion type nucleic acid(SSnA),triphosphare glyceraldehyde dehydrogenase(GAPDH) and so on. a new openreading frame was located between orf2 and mrp, The expression product of the new openreading frame had been confirmed to have IMPDH activity by our research group. IMPDHdomain was analyzed by Interpro and PHD software, and analysis revealed the consensuspattern was [LIVMT]-[RK]-[LIVM]-G-[LIVM]-G-X-G-[SRK]-[LIVMAT]-C-X-T.Cysteineresidue was thought to be involved in binding IMP.It was confermed the fusion expressionprotein of the open reading frame could extend the S stage of HEp-2 cell.1.Respective Fragment Deletion of IMPDH Gene of Streptococcus suisSerotype 2 and ExpressionIn the open reading frame encoding a polypeptide of 319 amino acid residues,noCysteine residue was found. According to the software analysis, IMPDH domain was locatedwithin the protein that was encoded by 9-798bp gene fragment. To investigate the catalyticsite and binding site of IMPDH, about 200 basepair was deleted from the 789 basepair everytime. According to the IMPDH gene of SS2 in GenBank, eight pairs of primers weredesigned, and the endonuclease of EcoRI, XhoI or NcoI was added to the end of eachprimer. Relevant gene fragments were connected by EcoRI, and were cloned into plasmidpET-32α(+). Five kinds of fusion protein were expressed,and the molecular weight of thefour kinds of deleted protein were 45.2kDa,43.8kDa,42.6kDa and 43.3kDa respectively, andthe total length protein was 52.2 kDa.2.Determination of The Functional Domain of IMPDH Gene ofStreptococcus suis Serotype 2Five kinds of fusion protein were expressed,and the five kinds of recombinanf protein containing six His-tag sequence were purified by its capacity of binding to Ni2+.Afterpurification by Ni-NTA His Bind Resin under native conditions, the five kinds of proteinwere detected in SDS-PAGE gel. By western blot experiment,the four kinds of deletedrecombinant protein were reactive with SS2 antiserum, and had different immunogenicreactivity with IMPDH monoclonal antibody. By IMP activity staining experiment, It wereconfirmed that the catalytic site and bindig site of IMPDH were located in the proteinencoded by 627-790bp gene fragment. Flow Cytometry(FCM) revealed that the five kindsof protein had different effect on Hep-2 cell cycle.