| Background: MgPais the main outer membrane protein of M.genitalium,which plays an important role in the adhesion and infection of M.genitalium to host cells.However,there areno reports on the specific binding of polypeptides,receptor peptides or receptor proteins to MgPa.M.genitaliumcan enter the host cell by cell phagocytosis and receptor mediate.Thus,blocking the cell receptor is an effective way to block the invasion of M.genitaliumand control M.genitalium’s infection.Therefore,the purpose of this study was to determine whether the specific binding of MgPa could inhibit the adhesion of MgPa and Mg to host cells and identify the receptors bound to MgPa from the host cells,whichpromote the understanding of the mechanism of M.genitalium infection,has important significance to the prevention and treatment of disease caused by M.genitalium infection.Objective : To investigate whether the MgPa-specific binding polypeptides selected in the previous study can inhibit the adhesion of rMgPa and Mg to host cells and provide a basis for elucidating the function of MgPa-specific binding polypeptides.MgPa cell receptors werescreened using modified VOPBA and the function was detected,which lay the foundation for effective prevention and treatment of Mg infection.Methods:(1)rMgPa,containing MgPa dominant epitope(1075~1364 aa),was expressed,purified by affinity chromatography and ultrafiltrated.The concentration was determined by BCA method.(V-H-W-D-F-R-Q-W-W-Q-P-S)(VS),(D-W-S-S-W-S-S-W-V-Y-R-D-P-Q-T)(DT)and(H-Y-I-D-F-R-W)(HW),which previouslyscreened from the phage random peptide library using rMgPa as the target molecule,were synthesized usingsolid-phase method.The indirect ELISA was used to detect the binding of rMgPa and synthetic peptides.The adhesion of rMgPa and Mg to SV-HUC-1 cells and whetherrMgPa antibody and synthetic peptide could inhibit the adhesion of r MgPa and Mg to SV-HUC-1 cells were identified by indirect immunofluorescence assay.(3)The receptors of MgPa on the SV-HUC-1 cell membranewere identified usingimproved VOPBA technique.The target proteins,contained in the target band,wereanalyzed byLC-MS.The specificity of receptor protein was analyzed by indirect ELISA and Far-western blotting.The distribution and function of receptor proteins on SV-HUC-1 cells were analyzed by indirect immunofluorescence assay.Results:(1)SDS-PAGE showed that rMgPa(containing 6 × His tag)with molecular weight of about 37 kDa and high purity was obtained and its concentration reached 2000 μg/mL.(2)The results of indirect ELISA showed that rMgPa could bind toVS,DT and HW,respectively,and could not bind specifically to the control peptide SP(S-V-S-V-G-M-K-P-S-P-R-P).(3)The results of indirect immunofluorescence assay showed that both rMgPa and Mg adhered to SV-HUC-1 cells,and that anti-rMgPa and three synthetic peptidescould inhibit the adhesion of rMgPa and Mg to SV-HUC-1 cells.(4)The protein with a molecular weight of about 17 kDa,from SV-HUC-1 cell membrane proteins,wasidentified using improved VOPBA technique,which can interact with rMgPa.(5)The results of LC-MS analysis showed that the potential receptor of rMgPa was Cyclophilin A(CyPA)and Histone H2B(H2B).(6)Indirect ELISA results showed that rMgPa could bind specifically to CyPA and H2 B protein.(7)Far-western blotting results showed that r MgPa could bind to CyPA and H2 B of SV-HUC-1 cell membrane protein;17kDa protein couldreact withthe specific CyPA antibody and H2 B antibody,which suggests17 kDa protein contains CyPA and H2 B.rMgPa could directly combine with CyPA and H2 B protein;(8)Indirect immunofluorescence assay showed that CyPA and H2 B were partially distributed on the surface of SV-HUC-1 cells.CyPA and H2 B could partially block the adhesion of rMgPa and Mg to SV-HUC-1 cells.Conclusions:(1)Three polypeptides screened from the phage random peptide library might be a potential receptor peptide of MgPa;(2)Cyclophilin Aand Histone H2 B may be the receptor proteins of MgPa on SV-HUC-1 cells. |
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