Screening Protective Antigens Of Erysipelothrix Rhusiopathia By Phage Display Combined With Local BLAST

Erysipelothrix erysipelas,whose pathogenic mechanism has not been clear,mainly cause swine erysipelas,which undoubtedly has made it difficult to prevent and control the bacterial infection.We used the phage display technology in combination with local BLAST to screen surface/secreted proteins of the Erysipelothrix erysipelas.The discovery of the surface/secreted proteins associated with virulence,often used as candidate antigens for the new vaccine,contributed to the Erysipelothrix erysipelas' pathogenic mechanism.The specific researches were as follows:We served anti-Erysipelothrix erysipela IgG as the target molecule and choosed two phage libraries(a loop-constrained heptapeptide library and a dodecapeptide library)to screen the B-cell mimotopes correlated with the Erysipelothrix erysipela surface.Then we excluded unrelated peptides(TUPs)from mimotopes through SAROTUP website.As a result,2743 mimotopes(1550 kinds of epitopes),including 33 kinds of TUPs,were obtained from the specific heptapeptide library;770 mimotopes(713 types of mimotopes),containing20 types of TUPs,were gained from the specific 12-mer peptide library.The correspondence between the B-cell mimotopes and the surface/secreted proteins of Erysipelothrix erysipelas were established by local BLAST.First,we used the subcellular localization websites to work out 334 possible surface/secreted proteins as a database to localize BLAST in the whole genome open reading frames of 3 Erysipelothrix erysipelas(serotype 1 and type 2);To verify the accuracy of the local BLAST,10 kinds of mimotopes were subjected to online and local BLAST analysis.As a result,the local BLAST outcomes of 3 mimotopes were not consistent with the online BLAST for that when a mimic epitope was conducted to online BLAST,it compared with the open reading frames of all strains of Erysipelothrix erysipelas,but its corresponding surface/secreted proteins were not conserved among all strains.The specific loop heptapeptides and dodecapeptides were subjected to local BLAST to determine their corresponding surface/secreted proteins.In consequence,1517 specific heptapeptides corresponded to 246 kinds of proteins with 209 mimotopes no corresponding proteins;693 specific dodecapeptides corresponded to 213 proteins with 29 mimotopes no corresponding proteins.A total of 269 kinds of surface/secreted proteins were screened by the two peptide libraries.In addition,the top 30 surface/secreted proteins corresponded to the two libraries respectively were consistent,which showed the screening results were credible.All mimotopes,corresponded to Spa A that is the mian protective antigen to induce anti-Erysipelothrix erysipela antibody,were returned to the Spa A's amino acid sequence.Consequently,the key amino acids of the Spa A's B-cell epitopes were in segment 19-35,49-54,61-68,81-84,121-128,150-155,196-199,231-237,256-260,280-291,303-308,311-314,325-331,354-365,377-380,408-411,417-420 and 438-443.Phage display technology combined with local BLAST could obtain quickly and efficiently large account of information about the Erysipelothrix erysipela surface proteins.The proteins with the high frequency,screened in both peptide libraries,had the potential as candidate antigens.And the possible B-cell epitope regions of a antigen could identified by returning its corresponded mimotopes to the amino acid sequence,which could lay the foundation for the study on pathogenes of the Erysipelothrix erysipelas and the vaccine development.