Immunoproteomics And Comparative Proteomics Of Streptococcus Suis Type 2

Streptococcus suis is a major swine pathogen with a worldwide distribution.Among the 35 serotypes described,SS2 is the serotype most frequently associated with a range of swine diseases including meningitis,pneumonia,septicemia,and arthritis.S. suis is also a zoonotic agent for people in contact with swine or their by-products, causing meningitis,permanent hearing loss,endocarditis,and septic shock[2,3]. From June to August of 2005,an outbreak of a severe invasive infection in humans and pigs occurred in Sichuan Province,China that resulted in 204 human infections and 38 deaths.This incidence followed a much smaller outbreak in 1998 in Jiangsu Province,which killed 14 of 25 patientsLacking suitable vaccine was one of the bottlenecks to control this infection.We describe our work using immunoproteomic approach,a method combining the specificity of antibody with precision of mass spectral analysis,to screen new antigenic proteins from extracellular proteins,membrane-associated proteins and cell-well proteins in SS2.The convalescent serum of a specific pathogen free(SPF) mini-pig recognized nine protein spots on PVDF membranes,which contained extracellurlar proteins of HA9801 and ZY05719 respectively.Antigenic proteins on a duplicate gel of ZY05719 and HA9801 were excised and identified by MALDI-TOF-MS.Peptide mass fingerprinting of the protein spots was performed using the MASCOT server.Sever novel antigenic proteins,as well as some knew antigenic proteins,MRP,EF and SLY, were identified.The membrane-associated proteins(MAP) were enriched using TritonⅩ-114 extraction protocol.These proteins were analyzed with two-dimensional gel electrophoresis(2-DE) and subsequent immunoblotting using hyperimmune serum of SS2-HA9801 immunized SPF mini-pigs.Thirteen antigenic proteins were recognized in the gels of MAP,and corresponding spots on each duplicate gel were excised and analyzed with MALDI-TOF-MS.cell-wall proteins of Streptococcus suis type 2 were extracted by mutanolysin protocol, then analyzed with two-dimensional gel electrophoresis(2-DE) and subsequent immunoblotting using hyperimmune serum of SS2-HA9801 immunized SPF mini-pigs.11 antigenic proteins were recognized in the gels of CWP,and corresponding spots on each duplicate gel were excised and analyzed with MALDI-TOF-MS.Seven immunogenic proteins were identified as significant by Mascot and 4 proteins were reported for the first time in SS2。Lacking suitable virulent markers was another bottleneck to control this infection. Comparative proteomic analysis was chosen to evaluate the differences of 2D gel profiles between two virulent strains(ZY05719 and HA9801) and an avirulent strain (T15).Eight of Nine differential proteins,which including MRP and EF,were successfully identified by MALDI-TOF-MS from the extracelluller proteins of SS2.Primers were synthesized against to the genes of identified proteins,PCR amplification and sequence revealed that genes for seven of the antigenic proteins,with five also in the avirulent strain,were found in both virulent strains and genes for six of differential proteins,with four also in the avirulent stain,were found in both virulent strains.Three differential proteins were also found in the bacterial proteins from the three strains of SS2.Primers were synthesized against to the genes of identified proteins,in order to find difference between virulent strains and avirulent strain at gene level.A immunogenic protein,named HM3,which was identified by using immunoproteomic assay.Partial gene of this protein were amplified form the genome of HA9801 by PCR and inserted into expression plasmid pET-32a(+) after double digested by BamHⅠand SalⅠ,then transformed into BL21(DE3) where they were induced to express by IPTG.After induced,there was specific proteins band of approximately 46kDa on the SDS-PAGE gel.Western-blotting showed that recombinant protein could react with immunized serum of HA9801 of SPF-minipig. This protein could take as a vaccine candidate of SS2。...