The Adhesive Characters Of P25 And P33 Proteins In Mycoplasma Gallisepticum S6 Strain

Mycoplasma gallisepticum(MG) is the primary causative agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys, which together cause significant economic losses in the poultry industry.MG is lack of cell wall, and mainly adhere to and infect the host cells by surface adhesion.Adhesion proteins are the key factors to locate in the surface of host cells,which play a significant role in MG pathogenic process. What’s more, adhesion proteins can be used as potential diagnostic antigens, which play a crucial role in the diagnostic application process of the MG’s infection. In this study we select a putative membrane protein gene p25 and a putative adhesion protein gene p33, mainly studying their possible prosperity of adhesion, laying the foundation for screening diagnostic antigens of MG S6 strain.This study abtained the in vitro expressed p25 and p33 gene by fixed point mutation, then got the soluble rP25/rP33, purified them to make anti-serum separately. Using anti-rP25/rP33 serum to identify MG S6 strain and Mycoplasma Synoviae(MS) respectively, and the result of western blot experiment showed that these two mycoplasmas can be well identifyed by anti-rP25/rP33 serum, thus we infer that P25 and P33 can be used as potential diagnostic antigens. The extraction of MG S6 membrane, cytoplasm protein and western blot showed that the distribution of P25 and P33 was in the surface of the membrane. Laser confocal microscope observed that both rP25 and rP33 can obviously adhere to DF-1 cells the same as MG S6. However, when rP25 or rP33 was incubated with rP25 or rP33 anti-serum, the adhesions were specifically inhabited and decreased significantly.The transmission electron microscopy and flow cytometry test also proved that the adhesion can be inhibited by the rP25 and rP33 anti-serum,what’s more, the combination of these two anti-serum can significantly inhibit the adhesion of MG S6 to DF-1 cells. So we proved that P25 and P33 are two adhesion related proteins of MG S6, and can be used as potential diagnostic antigens, which lay the foundation for the application research.In order to study whether it can cause related immune reaction after P25 or P33 adhere to DF-1 cells, we designed the qRT-PCR and ELISA assay to test the expression and release of cytokines. The results all showed both rP33 and rP25 can stimulate IL-1β、IL-2、IL-4、IL-10、IFN-γ to express and release in various degrees, thus we prove that P25 and P33 can serve as potential immune protective antigens.Above all, we proved that P25 and P33 are the adhesions of MG S6, and can be used as potential diagnostic antigens; In addition, we identifyed that these two proteins can induce DF-1cells to improve the expression of IL-1β、IL-2、IL-4、IL-10、IFN-γ, and can serve as potential immune protective antigens. All this can provide theory basis for the pathogenesis research of MG S6, development of new type of subunit vaccine, even the diagnosis, prevention and control of the infection of MG S6.