Screen And Indentification Of The Interacting Protein Of Mycoplasma Genitalium Protein Of Adhesion And Preliminary Study On Its Function

Background:Mycoplasma genitalium is the most minimal prokaryotic microorganism that lacks cell wall but has the ability to multiply in culture without life.The main adhesion protein（M.genitalium protein of adhesion,MgPa）is the key factor of M.genitalium adhering and invading to host.There were no reports on the study of MgPa interaction proteins at home and abroad and the biological function of Mg Pa was less studied.The interaction between M.genitalium and host cells is the key to the pathogenesis of M.genitalium.In this study,the phage display technique was used to screen and identify the interaction protein of MgPa from human urethral epithelial cells（SV-HUC-1）T7phage display cDNA library.The expression of differential proteins after the interaction between MgPa and RPL35 was studied by Tandem Mass Tags（TMT）labeling and proteome quantitative analysis,which provided the experimental basis for understanding the function of MgPa and further studying the pathogenic mechanism of M.genitalium.Objective:Screening and identifying the MgPa-interacting protein from SV-HUC-1 T7 phage display cDNA library,further study the effects of the interaction between MgPa and MgPa-interacting protein on cell function,to provide the experimental basis for understanding the biological function of MgPa and the pathogenic mechanism of M.genitalium.Methods:（1）Amplification of the SV-HUC-1 T7 phage display cDNA library.（2）Recombinant Mycoplasma genitalium protein of adhesion（rMgPa）was used as target molecule to biopan the T7 phage display cDNA library of SV-HUC-1 cell,positive clones were analysed using DNA sequencing and BLAST analysis.（3）Enzyme linked immunosorbent assay（ELISA）and dot immunoblotting test（dot-IBT）was used to identify the specific binding of positive phages to rMgPa.（4）Far-western blot was used to detect the specific binding of ribosomal protein L35（RPL35）to rMgPa.（5）The eukaryotic expression vector of Mg Pa（pcDNA3.1（+）/MgPa）was constructed and the interaction between RPL35 and MgPa was identified by indirect immunofluorescence（IF）.（6）Tandem Mass Tags（TMT）labeling and proteome quantitative analysis was used to screen the differential proteins before and after pcDNA3.1（+）/MgPa was transfected into SV-HUC-1 cells.（7）Real-time quantitative PCR（RT-qPCR）method was used to detect the expression of epidermal growth factor（EGF）,eukaryotic translation initiation factor 2A（EIF2）,signal recognition particle subunit SRP68（SRP68）,plasminogen activator inhibitor 1 RNA-binding protein（SERBP1）,60 S ribosomal protein L35a（RPL35A）,transforming growth factor beta-1-induced transcript 1 protein（TGF-β1）and cell proliferation was detected by MTT.Results:（1）The initial titer of phage cDNA library was 3x10⁸pfu after amplification.（2）After four rounds of affinity biopanning,the phage yield ranged from 0.67×10^-44 to 1.7×10^-3,the results showed that phages with specific binding to rMgPa was significantly enriched.（3）The results of BLAST analysis for PCR products showed that 32 positive clones randomly selected include 7 different sequences,which represent7 different proteins（P1-P7）,and the repetition rate was 62.5%for P1（RPL35）.（4）The results of indirect ELISA showed that all the 7bacteriophages could bind to rMgPa,and P1（RPL35）,P2（mitochondrio complete genome）and P3（RPN4）had strong binding force with the rMgPa.（5）Results of dot-IBT showed that obvious spots were appeared for P1,P2 and P3 representative phages,which demonstrated P1,P2 and P3 could combined specifically with rMgPa,these results were consistent with results of indirect ELISA.（6）The specific binding of RPL35 to rMgPa in total protein of SV-HUC-1 cells was further detected by Far-western blot.The results showed that there were obvious bands at about 37 KDa,indicating that RPL35 could specifically bind to rMgPa.（7）pcDNA3.1（+）/MgPa eukaryotic expression vector was constructed successfully that identificated by enzyme digestion and PCR.（8）M.genitalium could invade into SV-HUC-1 cells by the result of indirect immunofluorescence assay.（9）Results of Western blot and indirect immunofluorescence indicated that pcDNA3.1（+）/MgPa was successfully transfected into SV-HUC-1 cells.（10）RPL35 and MgPa can co-locate in SV-HUC-1 cells by indirect immunofluorescence analysis.（11）After pcDNA3.1（+）/MgPa was transfected into cells,the expression of 401 proteins were up-regulatedand 6 proteins were down-regulated by TMT analysis.（12）After pcDNA3.1（+）/MgPa was transfected into cells,the mRNA expression of EIF2,SRP68,SERBP1,RPL35 A,EGF and TGF-βwere increased in different degree by RT-qPCR（13）After pcDNA3.1（+）/MgPa was transfected into cells from24 to 48 hours,cell proliferation was obviously enhanced and inhibited after 72 hours by MTT assay.Conclusions:（1）The 60 s ribosomal protein L35（RPL35）may be the interaction protein of MgPa.（2）The interaction between Mg Pa and RPL35 can enhance the expression of transcription initiation and translation related protein,and promote cell proliferation.