The Screening And Identification Of Differential Proteins Between The Virulent And Attenuated Mycoplasma Hyopneumoniae Strain168

Mycoplasma hyopneumoniae (Mhp) was a major causative agent of Mycoplasma pneumonia of swine (MPS) which was a chronic respiratory disease with high infectious rate and low mortality. At present, some proteins related with Mhp virulence have been found, however, the differential protein of virulent and attenuated Mhp strain has rarely been reported.In this study, the proteins were extracted from the virulent and the attenuated Mhp strain168with the same source of the parental strain. Then the whole proteins were separated by two-dimensional electrophoresis (Two-dimensional Electrophoresis,2-DE),207±6protein spots were detected on the gel of the virulent strain, while153±4protein spots were displayed on the gel of the attenuated strain. By the comparison and the expression capacity analysis of the overall protein spots, a total of sixty-two difference points were found, of which fifty-two appeared on the map of the virulent strain, only three spots found on that of the attenuated virulent strain. And we also found that three protein spots were expressed higher in the virulent strain, while four protein spots were expressed higher in the attenuated strain. Twenty-four protein spots were analyzed by mass spectrometry, and nineteen spots were successfully identified. Of which there were four serum protein, four enzymes, a protein precursor, a hypothetical protein and an unknown protein products (may be not included in the protein database). By Western-blot analysis, the protein P36exhibits the better immunogenicity. In addition, according to the gene sequence of Mhp P36reported in GenBank, a pair of primer was designed. The expected gene was amplified by PCR, then inserted into expression vector pET-28a(+), and the recombinant plasmid pET-28a(+) P36was constructed. After it was successfully identified, the fusion protein was induced and expressed in E. coli BL21(DE3). When induced with1%IPTG after5h, the fusion protein expressed with the highest amount. The fusion protein was purified by Protino Ni-TED2000Packed Columns. Western-blot analysis indicated that the expressed protein have a very good reactogenicity. In this way, the fusion protein was hopeful to be used as a specific antigen for the establishment of Mhp ELISA detection.The differential proteins of virulent and attenuated Mhp strain168were analyzed and studied in immunity by using2-DE and Western-blot, it would be useful for laying the foundation of the pathogenic mechanism and prevention of Mhp for the further study.