Sdudy On Interaction Of MSG1Protein Of Mycoplasma Suis With Erythrocyte Membrane Proteins Of Porcine

Mycoplasma suis(M. suis) is the etiological agent of porcine eperythrozoonosis (PE). The pathogen is a kind of cell wall less and polymorphic microorganism that attach to the surface of erythrocytes and causes acute and sporadic infections. The acute clinical performances are high fever, anemia, jaundice and chronic infection are milder anemia, higher susceptibility to other infections and stresses. The disease are widely spreading in the world and leading to serious economic loss in the pig industry. However, M. suis cannot be cultured in vitro so far, and the pathogenic mechanism of M. suis infecting erythrocytes is not clear. The surface-located protein MSG1is an important virulence factor of M.suis which involved in the adhesion to erythrocytes. Since adhension to erythrocytes is indispensable in M. suis life cycle, it is very important to study the interaction of MSG1and erythrocyte membrane proteins. The main contents of the research are as following:1. Screening erythrocyte membrane proteins with recombinant Mycoplasma suis surface-localised protein MSG1.The porcine erythrocyte membrane proteins were extracted from M. suis negative pigs. Recombinant MSG1protein (rMSGl) fused with6×his was expressed in prokaryotic system and used to screen membrane proteins. MSG1recombinant protein and porcine erythrocyte membrane proteins were binding to the Ni-NTA agarose affinity column successively by affinity chromatography method. Erythrocyte membrane proteins binded to rMSGl were eluted after washing several time to exclude nonspecific binding to the column. SDS-PAGE analysis showed three candidate erythrocyte membrane proteins were obtained and mass spectrometry confirmed these three proteins were lactoferrin (LTF), β-actin and band3protein, respectively.2. Preparation and identification of polyclonal antibodies against erythrocyte membrane proteins.The2115bp LTF full-length gene was synthesized and used to amplify the N terminal 762bp fragment of LTF gene (LTF-N762). The purified gene were ligated into prokaryotic expression vector pET-32a(+) and transformed into E.coli BL21. SDS-PAGE analysis showed that the bacterial strains can effectively express recombinant protein. The polyclonal antibodies were prepared in mice and rabbits by back multy subcutaneous injection with purified recombinant proteins. The serum antibody titers were determined by indirect enzyme-linked immunosorbent assay (ELISA) after the third immunizations. The sera ELISA titers against LTF-N762were1:102400in mice and1:51200in rabbits, respectively. Western blot analysis showed the antiserums reacted with recombinant protein specifically.The rabbit polyclonal antibodies against band3protein was prepared with synthesized small peptide (band3:CGPEDPHIDYNQLGR) conjugated with KLH, which immunization and detection as above and antibody titers were1:512000. β-actin rabbit polyclonal antibody was commercialized. Western blot results showed these polyclonal antibodies could react with porcine erythrocyte membrane proteins specifically. Immunofluorescence assay (IFA) showed specific green fluorescence in the erythrocyte edge. The results demonstrated screened erythrocyte membrane proteins by affinity chromatography were all located on erythrocytes membrane.3. Analysis of M. suis MSG1protein interacting with erythrocyte membrane proteins.With purified M. suis MSG1protein and prepared monoclonal antibody against MSG1(1A7), immunofluorescence assay showed that rMSGl protein could attach to erythrocytes and mammalian cells specifically. MSG1protein co-located on erythrocytes with LTF, band3protein and β-actin by laser confocal assay. In addition, the specific interaction of rMSGl and β-actin was confirmed with siRNA interfering in mammalian cells.In summary, three porcine erythrocyte membrane proteins reacted with M.suis surface-located MSG1were screened in this study, and polyclonal antibodies against LTF and band3protein were prepared. This research further analysed the interaction of MSG1protein with LTF, band3protein and β-actin, which provided technical assist for the research on pathogenic mechanism of Mycoplasma suis.