| Mycoplasmal pneumonia of swine(MPS)is a chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae.Inactivated vaccination is currently the most important strategy for controlling the disease.However,these commercial vaccines can only provide partial protection and are not sufficient to eliminate pathogen from infected pigs.Furthermore,after immunization with inactivated vaccines,transmission of this respiratory pathogen is not significantly reduced.Investigation of new strategy and new candidate antigens is necessary for the development of next generation vaccines.The key issue for the development of vaccine is to screen antigens with good immunoprotective effect.Our laboratory has established the methods for screening the serological immunodominant proteins and screening the discriminative serological immunodominant proteins which can be recoganized by convalescent sera but not hyperimmune sera.These methods provide powerful tools to identify serological immunodominant proteins and discriminative serological immunodominant proteins in large-scale.In our study,we will optimize the ELISA method which was used to screen discriminative serological immunodominant proteins.Then we will screen serological immunodominant proteins and discriminative serological immunodominant proteins,providing candidate protein antigens for the screening of M.hyopneumoniae protective antigens.1.Optimization of ELISA method which was used to identify discriminative serological immunodominant proteins that can distinguish between hyperimmune sera and convalescent seraObjective: Optimizing the ELISA method which was to identify M.hyopneumoniae discriminative serological immunodominant proteins that can distinguish between hyperimmune sera and convalescent sera.Methods: The lysates of E.coli GST-Mhp366 and engineering bacteria E.coli GST were added into glutathione-coated 96-well microplates,and reacted with 9 hyperimmune sera and 15 convalescent sera,respectively.Then,the optimal working conditions,including antigen concentration,blocking buffer,and dilutions of serum and secondary antibody,were determined.Results: We could get the optimal antigen concentration,if the lysates of GST-Mhp366 recombinant bacteria and GST engineered bacteria were diluted 5 times.The optimal blocking buffer was 10% NBS and 2.5% skim milk dissolved in PBS.The optimal dilution of serum and HRP-conjugate rabbit anti-pig Ig G(H+L)secondary antibody were 1:500 and 1:40 000 diluted in blocking buffer.Conclusion: More precise working parameters were gotten for the ELISA method used to identify serological immunodominant proteins which can be identified by convalescent sera but not hyperimmune sera.Proteins identified by this method will be named discriminative discriminative serological immunodominant proteins.2.Prokaryotic expression of some M.hyopneumoniae protein antigens in soluble formObjective: Prokaryotic expression of some M.hyopneumoniae protein antigens in soluble form.Methods: Twenty-four ORFs were synthesized with the modification of TGA codons which encode tryptophan in M.hyopneumoniae genome to TGGs.Two ORFs,mhp067 and mhp565,were truncated into 5 fragments,respectively.Nucleotide fragments of mhp246-N,mhp246-C and mhp353-C were generated by PCR amplification from recombinant plasmids.Twenty four genes,namely mhp024,mhp067-1,mhp067-4,mhp067-5,mhp144,mhp170,mhp246-N,mhp274,mhp275,mhp303,mhp326,mhp347,mhp353-C,mhp354,mhp435,mhp440,mhp444,mhp460,mhp482,mhp520,mhp565-1,mhp565-2,mhp565-4,mhp565-5,mhp641 and mhp681 were cloned into p GEX-6P-1.Mhp246-C、mhp263 and mhp586 were ligated into p GEX-4T-3.Mhp067-3 and mhp320 were ligated into p GEX-5X-3.Mhp067-2,mhp565-3 and mhp586 were ligated into p GEX-6P-2.All recombinant plasmids were transformed into E.coli BL21(DE3)competent cells or E.coli XL-1 Blue competent cells,and the recombinant proteins were expressed by inducing.Crude purified recombinant proteins were cleaved with Pre Scission protease.Results: All 33 M.hyopneumoniae proteins were successfully expressed in soluble forms.Mhp067-4,Mhp144,Mhp275,Mhp520,Mhp565-2,Mhp565-5 and Mhp681 were cleaved off from the beads with Pre Scission protease.Conclusion: Thirty three M.hyopneumoniae proteins were successfully expressed in soluble forms.This provided materials for the identification of serological immunodominant proteins.3.Screening and identification of some serological immunodominant proteins of M.hyopneumoniaeObjective: Screening serological immunodominant proteins from 33 M.hyopneumoniae proteins.Methods: The lysates of 33 recombinant bacteria and engineering bacterium E.coli GST were added into the glutathione-coated plates and blocked with PBS+10% NBS+2.5% skim milk.Then,the proteins were reacted with sera(7 negative sera and 11 convalescent sera)with the dilution of 1:500.The plates were conjugated with 100 μl HRP-labeled rabbit anti-pig Ig G(Invitrogen,USA)diluted at 1:40 000 with blocking solution.Results: A total of 21 serological immunodominant proteins were identified,namely Mhp067-1,Mhp067-4,Mhp144,Mhp170,Mhp246-N,Mhp263,Mhp274,Mhp275,Mhp303,Mhp347,Mhp354,Mhp440,Mhp444,Mhp460,Mhp482,Mhp520,Mhp565-1,Mhp565-2,Mhp565-5,Mhp641 and Mhp681.Conclusion: Twenty one serological immunodominant proteins were screened from 33 M.hyopneumoniae proteins.4.Screening discriminative serological immunodominant proteins of M.hyopneumoniae which can discriminate between hyperimmune sera and convalescent seraObjective: Screening discriminative serological immunodominant proteins of M.hyopneumoniae which can discriminate between hyperimmune sera and convalescent sera From 21 serological immunodominant proteins.Methods: The lysates of 21 immunodominant protein recombinant bacteria and engineering bacterium E.coli GST with the dilution of 1:5 were added into the glutathione-coated plates and blocked with BS+10% NBS+2.5% skim milk.Then,the proteins were reacted with sera(7 negative sera and 11 convalescent sera)with the dilution of 1:500.The plates were conjugated with 100 μl HRP-labeled rabbit anti-pig Ig G(Invitrogen,USA)diluted at 1:40 000 with blocking solution.Results: A total of 6 discriminative serological immunodominant proteins were identified,namely Mhp067-4,Mhp170,Mhp275,Mhp347,Mhp444 and Mhp565-2.Conclusion: Six discriminative serological immunodominant proteins were screened from 21 serological immunodominant proteins of M.hyopneumoniae. |
PDF Full Text Request