Determination Of The Expression Of Fimbrial Subunit Protein A,C And E In Trueperella Pyogenes And Preparation Of Monoclonal Antibodies Against The Fimbrial Subunit Protein E

Trueperella pyogenes(T.pyogenes)is a commensal and an opportunistic pathogen in many different animal species.It's that mainly parasitizes the respiratory tract,digestive tract and reproductive tract of livestock and wild animals often causes inflamma tion in different tissues in a variety of animals.T.pyogenes fimbrial structure were also reported previously but few studies have been conducted on its structure and function.As one of the virulence factors of bacteria,fimbrial protein plays an important role in the process of bacteria attacking the host.Therefore,in-depth study of T.pyogenes fimbrial has positive significance for fully revealing the pathogenic mechanism and guiding the development of vaccine for T.pyogenes.In this study,the coding genes of T.pyogenes Fimbrial Subunit Protein(Fim),namely Fim A,Fim C and Fim E were cloned.Three recombinant Fim(r Fim)proteins,r Fim A,r Fim C and r Fim E were prepared.Three recombinant proteins were used to preparation rabbit polyclonal antibod ies.We demonstrated that only Fim E was expressed in cultured T.pyogenes with three polyclonal antibodies above.Western blot results showed that the expression of Fim E was not affected by anaerobic conditions,but was inhibited by microaerobic conditio ns and the expression level of Fim E reached the highest in the culture environment with p H 7.5 for 6~10 h.Tube agglutination tests indicated that Fim E was expressed on the surface of T.pyogenes because of strong agglutination with anti-r Fim E antibody.The fim C induce strong agglutination reaction with Anti-rfim C polyclonal antibody,but there was non-specific reaction by western blotting,therefore,it's undefined whether the expression of Fim C in T.pyogenes in this study.According to results of Tube agglutination test and Western blot,it was proved that the Fim A protein was not or poorly expressed in T.pyogenes under the culture conditions in this study.In this study,three monoclonal antibodies against protein Fim E were prepared by the cells hybridoma technique,named 2E6,16F7 and 3A5,and preparation of the ascites containing high concentrations of m Abs.Western blot results showed that all the three anti-fim E m Abs could react with r Fim E,and protein with molecular weight of 58 kDa in T.pyogenes lysates specifically,which was consistent with the predicted molecular weight of natural Fim E protein.Dot-elisa results showed that the three m Abs only reacted with T.pyogenes bacteria lysates,but did not react with T.pyogenes culture supernatant and e.coli lysates.Prepared m Abs could specificly react with T.pyogenes Fim E relatively,according the results of above.The response characteristics of the three m Abs were further analyzed,and the results showed that the coding region of the epitope was in the coding region of 189-277 aa and 438-511 aa of the Fim E.In conclusion,our study confirmed that Fim E could be expressed stably during the T.pyogenes cultured,and anti-fim E m Abs were prepared,which provided a theoretical basis and material basis for further study on the structure and function of T.pyogenes fimbrial.