Analysis Of The Major Outer Membrane Immunological Protein Of Mycoplasma Bovis And Establishment Of Indirect ELISA For Detecting Antibody Of Mycoplasma Bovis

The small wall-less prokaryotic species Mycoplasma bovis is a major pathogen that causes mastitis, arthritis, pneumonia, as well as diseases of the genital tract in cattle and considerable economoic losses in cattle industry. There are currently no vaccines or diagnostic tools available. The antigen repertoire of this pathogen includes a family of variable surface lipoproteins (Vsps) which represents a set of immunodominant lipoproteins undergoing high-frequency phase and size variations, has important signifieanee for Mycoplasma bovis infection diagnosis and vaccine development.1Estraction and antigencity of Mycoplasma bovis VspThe outer membrane proteins of Mycoplasma bovis were estracted through Tritonx-114lysis, Tritonx-100and KI lysis respectively. Two protein bands were chosen for LTQ mass spectrometry identification according to SDS-PAGE and western blot results.The molecular weight of protein was identified to range from25to95KD by SDS-PAGE,and the western blot result indicated that the four protein bands (40KD、51KD、69KD and72KD) are the main protection antigen.It was also found that Tritonx-114was more effective than Tritonx-100、KI for extracting outer membrane proteins from Mycoplasma bovis.Two membrane proteins80、48and mlecular chaperone DnaK of21proteins identificated by LTQ mass spectrometry identification could be candidates for vaccine development. 2Prokaryotic Expression and purification of MB-Vsp repeating peptide fragmentThe MB-Vsp repeating peptide fragment which selected by biological informatic analysis was cloned to plasmid vector pET-32a(+), and the combinant plasmid was expressed in the E.coli BL21,the purified fusion protein showed good immune reactivity detected by western-blot.3Establishment and optimization of the Indirect ELISA method for detecting antibodyThe indirect ELISA method for the detection of the mycoplasma bovis antibody was established using the purified recombinant protein as coating antigen. Through repeatedly test and confirmed the optimization test conditions:the coating concentration of antigen was10.28μg/mL, the optimum dilution of the tested serum should be1:100,the sealed buffer was5%coat serum and37℃blocking for60min,the action between antigen and antibody lasted for30min,the optimum actioned period for the binding of antigen with enzyme-signed SPA which were diluted by1:20000was30min, samples with S/P>0.494were scored positive base on the ELISA test.In addition, repeatability tests revealed that the coefficient of variation of positive sera within and between runs were2.802～8.179%and7.573～1.0.283%.We used the indirect ELISA developed by us to detected mycoplasma agalactiae、Bovine viral diarrhea (BVD)、Foot and Mouth Disease (FMD)、Bovine infectious rhinotracheitis positive serum,the result were negative.It proved that indirect ELISA established in this study has good specificity.4The comparison of four indirect ELISA methodsTo compare the indirect ELISA developed by us with there commercial kits from Canada, Belgium and Harbin Veterinary Research Institute, seventy sera samples were detected by the four methods. The correlation rate between the indirect ELISA estabilished in this study and ELISA kit from Canada was92.86%, the correlation rate between the indirect ELISA estabilished in this study and ELISA kit from Belgium was82.85%,and the correlation rate between the indirect ELISA estabilished in this study and ELISA kit from Harbin Veterinary Research Institute was95.71%; Moreover, test on the positive serum of mycoplasma agalactiae, there commercial kits showed positive detection results, but it was negative by the indirect ELISA developed by us. The results showed that the method developed with the recombinant are sensitive、specific and can differentiated mycoplasma bovis and mycoplasma agalactiae.In conclusion, the outer membrane proteins extracted from Mycoplasma bovis showed good immune reactivity detected by western-blot, and there membrane proteins were selected for candidates of vaccine according to SDS-PAGE、westem blot and LTQ analysis. The indirect ELISA method for the detection of the mycoplasma bovis antibody with high sensitivity、specificity、and repeatability was established using the purified recombinant protein as coating antigen,which laid the foundation for differential diagnostic ELISA Kit of Mycoplasma bovis.