Prokaryotic Expression And Immunogenicity Assay Of Mycoplasma Hyopneumoniae Membrane Proteins

Mycoplasmal pneumonia of swine （MPS） is known as enzootic pneumonia （EP）, a chronic respiratory disease caused by Mycoplasma hyopneumoniae （MHP）. The main symptoms of MPS are coughing and gasping. The infected pigs are easily infected by other pathogens, which may increase the difficulty to control swine diseases. Moreover, MPS may cause poor feed conversion and retarded growth of pigs, and lead to considerable economic losses in the swine industry. Vaccination is one of the effective ways to control MPS. At present, conventional inactivated vaccine and attenuated vaccine are widely used in prevention of MPS. However, M. hyopneumoniae must be cultured under strict condition and its incubation period is longer than general bacteria, increasing the cost of production of inactivated vaccine and attenuated vaccine. Therefore, It is urgent to develop safer, more effective and inexpensive new vaccine against MPS. The identification of novel immunogenic antigens is an important step for development of M. hyopneumoniae vaccines. Cell membrane of M. hyopneumoniae plays an important role in its pathogenic process. The function of lots of membrane proteins is still unknown. In this study, we evaluated the immunological characterization of34membrane proteins of M. hyopneumoniae, aiming at exploring novel immunological proteins to provide candidates for the development of subunit vaccine and diagnostic methods. The main studies are as following:1. Selection and cloning of candidate protein genesThe whole695CDS of M. hyopneumoniae strain168and currently known immunogen proteins of mycoplasma were analyzed by bioinformatics software （TMHMM）.34previously uncharacterized CDS, witch encode predicted membrane proteins with single transmembrane region and the transmembrane region located in the N-termimal were selected.2. Expression of recombinant proteinsAmong the34CDS, there were30CDS using TGA encoding tryptophan. Codon TGAs encoding tryptophan in M. hyopneumoniae were mutated into TGGs encoding tryptophan in E. coli by SOE-PCR. Then the30amplified fragments and another4CDS amplified by ordinary PCR were successfully purified and cloned into pET-28a expression vector. The34recombinant expression plasmids were transformed into E. coli BL21（DE3） or Rosetta （DE3） competent cells. IPTG was used to induce the expression of6His fusion proteins. From the34fragment cloned, expression of26recombinant proteins were detected by SDS-PAGE.3. Reactogenicity analysis of candidate proteinsSitu Colony Blot and Western Blot of recombinant bacteria were used to roughly screen the34candidate proteins against MHP incubated pig serum.6recombinant bacteria were recognized by positive serum from MHP infected pigs. The proteins are MHP168₃22, MHP1688₄7, MHP168₃48, MHP168₃98, MHP168₆22and MHP168₆39, respectively. Furthermore, candidate proteins MHP168₃47, MHP168₃48, MHP168₆22, MHP168₆39and positive control P46protein were purified as antigen. Serum from MHP infected pigs and inactivated vaccine or attenuated vaccine vaccinated pigs were taken as antibody. The indirect ELISA results show that proteins MHP168₃47, MHP168₆39, MHP168₃48and positive control P46significantly reacted with serum from MHP infected pigs compared with serum from MHP negative pig. And MHP168₆22can strongly react with both positive serum and negative serum, suggested it is not a MHP specific protein. Moreover, MHP168₃47, MHP168639, MHP168348and P46can react with serum from attenuated vaccine vaccinated pigs significantly higher compared with PBS control. And MHP168₃48were slightly higher than P46, indicated that MHP168₃48may be an important protective antigen. But the3candidate proteins and P46protein did not react with serum inactivated vaccine inoculated pigs.4. Immunogenicity analysis of the candidate proteins in micePurified candidate proteins MHP168₃47, MHP168₃48, MHP168₆22and MHP168₆39were inoculated in female BALB/c mice subcutaneously. Protein P46and PBS were taken as positive control and negative control, respectively. Purified candidate proteins and3strains Mycoplasma hyopneumoniae cell lysate were taken as antigen by indirect ELISA and Western Blot to evaluate the level of humoral immunity in mice inoculated with candidate proteins. The results show that MHP168₆39, MHP168₃47and P46induced much higher levels of antibody than PBS. But only MHP168₆39and P46can react with M. hyopneumoniae cell lysate, indicated that the3strain MHP may express MHP168₆39in vitro.Summary, the immunogenicity of34membrane proteins of M. hyopneumoniae which had never been characterized before were analyzed in this study. Candidate proteins MHP168₃47, MHP168₃48, MHP168₃98and MHP168₆39might be immunodominant protein of M.hyopneumoniae. Our study provided a foundation for the development of subunit vaccines and establishment of diagnostic methods.