Characteristic Identification Of Mycoplasma Gallisepticum Adhesin Protein And The Distribution Of Its Interactive Proteins In Chick Embryo Tissues

Mycoplasma gallisepticum(MG) is the etiologic agents of chronic respiratory disease which commonly occurred in the world and resulted in severe lose to poultry industry. Researches had indicated that MG infested host by connecting its protein of Mycoplasma Gallisepticum Adhesin(pMGA) with surface receptors on the mucosa of respiratory tract. To eliminate pMGA structure,function and molecular pathogenic mechanism of MG,the characteristics of pMGA was identified,and the interaction of pMGA with receptor in host was discussed.Moreover,receptor distribution in chick tissues was detected.These results including below,will be helpful to research the genotype,molecular structure and acting mode of pMGA receptor.1.Characteristic Identification of Mycoplasma Gallisepticum Adhesin ProteinBased on amino acid sequence,bioinformatics analysis of pMGA physical and chemical characteristics,domain,phosphorylated sites,sequent and space structure were carried out with biological software and online analytical tool.pMGA1.2 bio-function mainly occurred out of cell.Its molecular weight was 64.9kD,isoelectric point in abstracto was 5.58,estimated half-life were 30 hours,20 hours and 10 hours respectively in mammal reticulocyte,yeast and E.coli.pGMA1.2 was stable globular protein because of that its secondary structure had 26.5%a-helix mainly in front of the 210 amino acids, 23.08%βfolds and 50.33%andom coil mainly behind the 210 amino acids.There were 6 motif modes,53 motif sites,21 antigen determinants and 4 obvious coiled coils in pMGA1.2,pGMA1.2 also included a typical domain of the mycoplasma hemaglutinin superfamily,two FIVAR domains(Uncharacterized Sugar-binding Domain),one domain of methylated DNA-protein cysteine methyltransferase of C-terminal,one galactose binding domain-like.pGMA was also a member of MG hemagglutinin superfamily and had 99%semblance with adhesin protein sequence in R strain mycoplasma.2.Immunological Characteristics of Expression Product of Brachytmema and Complete Proteins of Mycoplasma Gallisepticum AdhesinUnder the optimum conditions,the soluble expression quantities of pMGA1.2 were highly to 18.5%and 17.0%in complete thaline and supernatant respectively,and expression amount of pMGAⅣwere up to 28%and 18%respectively in complete thaline and supematant.95%purity of pMGA1.2 and pMGAⅣwere obtained with GST affinity column.Obviously colored straps emerged with immunoblotting while fusion proteins of pMGA1.2 and pMGAⅣreacted with specific positive sera of MG,demonstrated that fusion proteins of pMGA1.2 and pMGAⅣhad satisfactory Immunologic competence.3.Receptor Distribution of pMGA in Chick Embryo Tissues with ImmunofluorescenceIndirect immunofluorescent histochemistry test was set up with purified pMGA1.2 separated from pMGA1.2·GST,in which the GST was cut off with Thrombin.Paraffin sections of tissues were respectively reacted with pGMA1.2,MG sera from rabbits and secondary antibodies marked with FITC in proper order.Beside spleen,specific fluorescence could be observed in trachea,lung,liver,kidney,duodenum,bursa of Fabricius,glandular stomach,thymus and heart from twenty days old SPF chick embryo, which suggested that specific pMGA1.2 receptors could reside in all these nine tissues or organs.Moreover,pMGA receptors mainly located on lymphatic nodule cortical area of diverticule of bursa of Fabricius,thymic cortical part,mucosa of glandular stomach,and ciliated epithelial cells of trachea,bronchus and duodenum.However,the distributed site of pMGA receptor in liver,heart and kidney couldn't be defined still.4.Specific Combination of pMGA with Membrane Proteins in Chick Embryo TissuesMembrane proteins was abstracted with differential centrifugation,and doted on nitrocellulose filter.As target proteins,purified pMGA1.2 and pMGAⅣwere connected with membrane proteins and antibodies of MG from rabbits,and MG antibodies were combinated with IgG marked with HRP.SDS-PAGE was also applied to identify receptor proteins.The results showed that the membrane proteins with molecular weights 36.5kD and/or 27.2kD,specifically binding with pMGA1.2 and pMGAⅣ,might be receptor proteins of pMGA1.2,but pMGAⅣwas a peptide connecting receptor proteins and pMGA1.2.