Development Of ICS For Antibody Detection Of S. Suis 2 And The Protein-protein Interaction Between FBPS And The Host

Streptococcus suis (S. suis) is a common pathogenic microorganism and can cause avariety of clinical diseases in human and swine. Based on the CPS antigens of S. suis, 35serotypes or capsular types have been characterized. S. suis serotype 2 (SS2) is mostcommonly associated with severe disease and is the serotype most frequently isolatedfrom pigs.S. suis is an opportunistic bacterium of pigs. Crowding, poor ventilation, suddenweather changes, mixing, relocation, immunization and the presence of concurrentinfections could predispose pigs to SS2 infection and morbidity. Thereforesero-surveillance or antibody monitoring may be useful in determining the infection statusand can play an important role in the control Streptococcosis in pigs.In this study, an immunochromatographic strip (ICS) was developed for thedetection of antibody against SS2. Colloidal gold particles labeled with staphylococcalprotein A (SPA) were used as the. detector reagent. The capsular polysaccharide (CPS) ofSS2 and affinity purified IgG from a healthy naive pig were immobilized on test andcontrol regions of a nitrocellulose membrane, respectively. An Enzyme linkedimmunosorbent assay (ELISA) for SS2 antibody detection based on purified CPS wasused as a control method.The ICS was used to (i) detect anti-CPS antibody in 16 sera taken from 4 SS2infected pigs, 24 sera from pigs hyper-immunized with SS2 and 68 sera from pigsinoculated or infected with bacteria other than SS2; (ⅱ) determine anti-CPS antibodytiters of 20 positive sera for comparison with ELISA; (ⅲ) detect anti-CPS antibody in 486clinical sera taken from diseased pigs also for comparison with ELISA.For 16 sera from pigs that had survived challenge with SS2, both ICS and ELISAgave all samples positive results. For the 24 hyper-immune sera raised against SS2, therewas also a 100% correlation between ELISA and ICS results. All of the 68 sera againstnon-SS2 bacteria, except one antisera against S. suis type 1 determined as weak positiveby both ICS and ELISA, were negative in both ELISA and ICS tests. Antibody titers of 20hyper-immune sera raised against SS2 were tested by ICS and ELISA. The quantifyingtest result indicated that the sensitivity of the strip test is close to or slightly less than thatof ELISA.Of the 486 clinical sera from sick pigs, 228 were antibody positive and 258 werenegative by ELISA, whereas 223 were positive and 263 were negative by ICS. Additionally 209 were positive in both assays and 244 were negative in both tests. Thus,the specificity and sensitivity of ICS, compared to ELISA, was 96.4% and 91.7%,respectively. There was an excellent agreement (kappa=0.863) between ICS and ELISA.Although some assays, e.g. the indirect ELISA and agar gel precipitation tests, havebeen developed for antibody detection for S. suis, these methods are not suitable for useoutside of the research laboratory. In contrast, the ICS is easy to use and rapid. In thiscontext, the ICS can be used as a tool for seroepidemiological survey of pigs fromdifferent areas in China. This would in turn facilitate the formulation of effective controlmeasures.To understand the pathogenesis of S. suis infection contributes to develop strategiesfor prevention or therapy. The interactions between pathogenic microorganism and hostcells may play an important role in its pathogenicity. In this study, the CytoTraptwo-hybrid syste was used to investigate the protein-protein interaction between FBPSand the host cells.FBPS, which can bind to human FN and FGN, is a protein coded by Fbps gene of S.suis. And Fbps gene is present in all known serotypes of S. suis except serotype 32 andserotype 34. A considerable number of FN and FGN binding proteins of various bacterialspecies have been described. Most of these proteins have been shown to be involved inadhesion to epithelial and/or endothelial cells. FBPS play a role in the SS2 colonization ofspecific organs.The full length Fbps gene was amplified from chromosomal DNA of SS2 strainSCZY05 by PCR. Fbps gene was unidirectional cloned into pSos after the amplified PCRproduct and pSos vector were digested with BamHⅠand NotⅠ, yielding the bait plasmidpSos-Fbps. The recombinant expression plasmid pET-28c-Fbps was constructed by thesame way.The library was screened by co-transformation of the pSos-Fbps bait construct into atemperature-sensitive cdc25H yeast strain that can not grow at 37℃. If the bait proteinphysically interacts with the target protein, the hSos protein is recruited to the membraneactivating the Ras signaling pathway and allowing the cdc25H yeast strain to grow at 37℃. The clones growing at 37℃on SD/galactose (-UL) plates but not on SD/glucose(-UL) plates were selected as "putative positive" clones and were analyzed further.The Target plasmid DNA was isolated from pMyr eDNA putative positive clones andwas transformed into E. coli cells for plasmid amplification. Colonies contains eDNAfragment insertion identified by restriction digest analysis. The positive Target plasmid was sequenced on a 3730 Sequencing system. The sequencing results were sent to NCBIfor BLAST searches on line. In this study, we got two positive target plasmid. One ofthese genes is identical to the apoptosis inhibitor 5 (API 5) gene. The other showedhomology to genes encoding zinc finger FYVE domain containing 27 (ZFYVE 27).The protein-protein interaction between FBPS and API 5 or ZFYVE 27 wasconfirmed by ProFound Co-immunoprecipation assay. Recombinant plasmid pET-28c-Fbps was transformed into E. coli BL21 cells for protein expression. The full length ofZFYVE 27 and API 5, obtained by PCR from the target vectors were subcloned intopcDNA3.1/myc-His, yielding pcDNA3.1/myc-His-ZFYVE 27 and pcDNA3.1/myc-His-API 5. The recombinant expression plasmids were transfected into 293T cells for proteinexpression. Anti-mye probe antibody was used to immunopreeipitate the expressedprotein (API 5 or ZFYVE 27) from the whole cell lysate and supernatant of BL21transformed with pET-28c-Fbps culture. The complex was then resolved on a 10% SDSpolyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane.Pig anti-sera against SS2 were used for western-blotting.