| 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 serotype2(SS2) is mostcommonly and most virulent of these serotypes.Glutamate dehydrogenase (GDH) is a critical enzyme for connection between carbon andnitrogen metabolism in S.suis. It is also a very important function product in the course ofenergy metabolism in bacteria and is critical of bacterial pathogenicity. The GDH protein of S.suis is a highly conserved genera-specific antigen (MW of about48.8kDa), which exposed onthe surface of S. suis cells and can be used as an important antigen to establish a generalmethod for diagnosis of S. suis.In present study the GDH gene of S.suis strain of SC22was cloned into prokaryoticexpression vector pET-30(a), expressed in E. coli after IPTG induction. Expressed fusionprotein was purified by Ni-NTA, and its biological activities were measured by animalexperiment afterwards. Based on the NC strip and plate two indirect ELISAs for the detectionof antibody against S.suis was developed by using the recombinant expressed GlutamateDehydrogenase (rGDH) protein.1. Prokaryotic expression of gene encoding glutamate dehydrogenase of SS2andpreparation of polyclonal antibodies against its expressed productsTo obtain detection antigen for diagnosis of S. suis infection. The complete ORF ofglutamate dehydrogenase (GDH) gene was amplified from the genomic DNA of SS2strainSC22isolated in Sichuan Province by polymerase chain reaction (PCR). The resultingproduct was cloned into the prokaryotic expression vector pET-30a, which was thentransformed into E. coli BL21(DE3). The identified positive transformants were screened for expression induced by IPTG. The expression products were subjected to SDS-PAGE and therecombinant protein was purified by nickel ion-agarose affinity chromatography. NewZealand rabbits were immunized with the purified recombinant GDH protein to preparepolyclonal antibodies. Titers of the anti-serum were determined by indirect ELISA andWestern blot assay. The recombinant GDH protein was effectively expressed in the hostbacteria, and highly pure recombinant protein was obtained by nickel ion-agarose affinitychromatography. High-titer anti-serum against the recombinant protein was obtained. Asevidenced by western blot assay, the sera could react specifically with the lysates of alldetected S. suis strains. In addition, the recombinant GDH protein could react specificallywith serum samples collected from five pigs experimentally infected by strain SC22. Theexpressed GDH fusion protein has some common epitopes of natural GDH and can be used asdetection antigen to develop ELISA and other diagnostic methods．2. Development and application of PPA-ELISA for detection of antibodies against S. suisusing recombinant GDH protein as antigenAn indirect ELISA was developed for the detection of specific antibody against S. suis byusing the reombinant expressed glutamate dehydrogenase (rGDH) protein as a coating antigen,horseradish peroxidase labeled SPA (PPA) as the second antibody. The optimal concentrationof coating recombinant antigen was12.5ug/mL and the optimal dilution of serum sample was1:80in the cross assay. PPA was used at1:4000dilution. The cut-off was chosen as an0D450≥0.378for positive respons. The specificity test indicated that no cross-reactionoccurred between antibodies against common diseases of swine. The variation coefficient ofintra-batch and the inter-batch in the repeating tests was less than10%. After vaccination andchallenge, serum samples were collected periodically for detecting titer. The geometricaverage (GMT) of titers was calculated, and the curve of titer was drawn for each test group.Using the PPA-EIISA and commercially successful ELISA kit,120serum samples wereparallelly tested, and the coincidence rates were96.7%. A total of320sera were detected and73.3%of them were positive. Experiments confirm that the PAA-ELISA is a convenient, rapid,more sensitive and specific diagnostic method and was provid a new tool for the large-scaleepidemiological surveys and serological diagnosis of streptococcal infection. 3. Development and application of Dot-PAA-ELISA rapidly detecting antibody byprokaryotic recombinant expression product of S. suis GDH geneBased on the NCM strip, an indirect Dot-PPA-ELISA for the detection of antibodyagainst S. suis was developed by using the rGDH protein. The rGDH coated antigen was usedat an optimal concentration of12.5ug/mL and PPA was used at1:2000dilution. The optimumcondition for the closure is37℃for1h. Thirty serum samples from growing rabbits fromvarious geographical locations were measured by Dot-PPA-ELISA to determine the antibodieanginst SS. The comparable result up to97.5%agreement, was obtained between our newDot-PPA-ELISA and the normal ELISA tests carried on the microtiter plate by simultaneoudetecting120samples. A total of320sera were detected and73.3%of them were positive.The results showed that the Dot-PAA-ELISA is a convenient, rapid, more sensitive andspecific diagnostic method and could detect antibodies against all serotypes of S. suis andmay provide a simple and rapid method for seroepedwmiological surveys of S. suis infection. |
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