Preparation Of Monoclonal Antibodies Against IMPDH In SS2 And Analysis Of Its Epitopes

Streptococcus suis serotype 2 is now recognized as the causal agent of swine streptococcus suis in our country.It causes meningitis,arthritis,endocarditis,sepsis, pneumonia,and sudden death in young pigs.It is an important zoonotic disease pathogen that can infect and cause death on associated people.SS2 can be divided into virulent strain,low virulent strain and avirulent strain.There is a new open reading frame between ORF2 and MRP.New ORF was confirmed possessing the activity of inosine monophosphate dehydrogenase(IMPDH) by activity straining in our research group.In order to learn more about the IMPDH's immunological characteristics of SS₂,the monoclonal antibodies against IMPDH protein of SS₂ were prepared by the purified IMPDH fusion protein,and analysed its epitopes.1.Preparation and identification of monoclonal antibody against IMPDH protein of Streptococcus suis Serotype 2The gene of IMPDH was amplified by PCR and cloned into prokaryotic expression vector pET32a.The positive plasrnid contained IMPDH gene was determinded by restriction enzyme analysis,then transformed into E.coLi BL21 cells.A fusion protein which was about 48kDa was expressed after BL21 mentioned above was induced by IPTG. Spleen cells from BALB/c mice immunized with purified fusion protein were fused with SP2/0 myeloma cells after the last immunization.Hybridoma supemants were screened for the specific antibodies by indirect enzyme-linked immunosorbent assay(ELISA).After four times of limiting dilution assay,four strains of hybridoma cell(1A8,1F2,2D2,2D12) were successfully obtained,which can be cultured stably and secreted monoclonal antibody (McAb) against IMPDH.The ELISA titers of ascites were 100×2¹¹,100×2¹¹,100×2¹⁰ and 100×2⁸.The analysis of Western-blot indicated that the four strains of monoclonal antibody can react with IMPDH and not react with TrxA.2.Analysis of differences in epitopes of the McAbsFour fusion proteins were constructed respectively by the means of deleting four different fragements of IMPDH gene.The differential recognition of epitopes was studied by western-blot in which the four fusion proteins were used as antigens.The results indicated that 1A8,1F2,2D2 possess the specificity of reacting with P-D1,P-D2,P-D3 fusion protein and 2D12 possess the specificity of reacting with P-D1,P-D2 fusion protein.Initially inferred that 1A8,1F2 and 2D2's epitopes concentrated on the site of 627-790bp of IMPDH gene,otherwise,2D12's concentrated on the site of 411-790bp. Then,the two fragments(627-790bp,411-790bp) were amplified by PCR and inserted into prokaryotic expression vector in order to get the objective proteins.The results of western-blot showed that 1A8,1F2,2D2 can react with the products of 411-790bp gene and 613-809bp gene.However,2D12 can only react with the product of 411-790bp gene but not with the product of 613-809bp gene.The facts quoted above fully confirmed what was concluded in preliminary experiments,which also consummated the recognition of IMPDH's immunological characteristics in SS2 and inferred the two epitopes belong to linear epitope.