Preparation And Evaluation Of The Immunoprotective Effect Of Recombinant Immunogens Based On The Antigenic Dominant Region Of Pyolysin

Trueperella pyogenes(T.pyogenes)is an important opportunistic pathogen,and always be found in the mucous membranes of cattle,sheep,pigs and other important economic animals.T.pyogenes can spread widely among domestic animals,resulting in a variety of non-specific purulent infections of the skin,internal organs and joints,bring greater economic losses.Plolysin(PLO)is the only hemolysin secreted by T.pyogenes,and it is the primary virulence factor of T.pyogenes.PLO can be obtained from all T.pyogenes strains currently,and it has strong immunogenicity.In this study,anti-rPLO polyclonal antibodies in different animals were prepared using recombinant PLO(rPLO),and the response of five segments of PLO Domain 1-3(PLO D123)truncated proteins rPLO 1-110,rPLO 95-156,rPLO 141-204,rPLO 190-296,rPLO 281-393 was detected by different animal serums.Western-blot results showed that rabbit anti-rPLO polyclonal antibody can only react with rPLO 1-110 and rPLO 190-296 specifically,and the mouse anti-rPLO polyclonal antibody can react specifically with rPLO 1-110,rPLO 95-156 and rPLO 190-296.The chicken anti-rPLO polyclonal antibody reacts with four truncated fragments except rPLO 281-393.It is worth noting that the truncated fragment rPLO 281-393 did not react with any animal anti-PLO polyclonal antibody.Therefore,we infered that PLO 281-393 is an immuno-non-dominant region.Since anti-rPLO polyclonal antibodies of three animals can react with rPLO 1-110 and rPLO 190-296.Therefore,to further determine the epitopes in PLO 1-110 and PLO 190-296.In this study,rabbit polyclonal antibody against rPLO were used to screen the linear epitopes of B-cells Trx-EP1(rPLO 20-33)and Trx-EP2(rPLO 249-260)in PLO D123.It was found that the chicken anti-rPLO polyclonal antibody and the mouse anti-rPLO polyclonal antibody can react with four adjacents truncated peptides with overlapping parts of Trx-EP1 and Trx-EP2.This shows that the two regions EP1 and EP2 are immunodominant and may be located outside the PLO molecule.In addition,polyclonal anti-EP1 and EP2 epitope peptides were prepared in this study,and perform anti-hemolytic activity analysis,it was found that the mouse anti-Trx-EP2 polyclonal antibody can neutralize hemolysis caused by rPLO,whereas chicken anti-Trx-EP2 does not.This shows that specific antibodies against the same epitope may have different results depending on the experimental animals.On this basis,the two antigen-dominant regions in PLO D123 were fused to PLO D4,respectively.Recombinant antigen proteins rPLO 1-110-D4 and rPLO 190-296-D4 were prepared.The experimental animals were immunized with red blood cell membranes as adjuvant or Freund's adjuvant mixed with recombinant proteins.The results of the challenge experiment show that,mouse immunized with rPLO 1-110-D4 and rPLO 190-296-D4 as vaccine antigens could give mouse some resistance to T.pyogenes challenge.In summary,the antigen-dominant regions of PLO D123 were identified in this study and constructed two recombinant antigens.The challenge protection experiment showed that mouse immunized with the recombinant antigens can prolong the survival times of mouse after bacterial attack and improve the survival rate.It provides animals with incomplete immunity against T.pyogenes infection.This study provides a new basis for the study of the structure and function of PLO,and it also provides new ideas for the development of novel vaccines for T.pyogenes.