Screening And Identification Of The Mycoplasma Gallisepticum Glyceraldehyde-3-phosphate Dehydrogenase Protein As A Pretective Antigen Candidate

Mycoplasma gallisepticum(MG) is the main pathogen of chronic respiratory disease in chickens and other poultry, and it is also one of the common pathogens in the modern poultry industry, which causes significant economic loss in all phases of the poultry industry and often coinfects with other virus or microbiologies. Though inactivated-MG vaccines or live attenuated-MG vaccines are often used to efficiently prevent MG infection. The two kinds of vaccines are insufficient as high cost or a risk of virulence return and infection of non target chicken and need to be improved. The development of more economic, safe and efficient vaccines is applauded for prevention and control of MG, in which suitable candidate antigen is so important but difficult. In this study, based on the fact that the local (mucosal) specific antibody is particularly important in MG infection, we collected the respiratory tract washings (RTWs) of MG-infected chickens and used them to screen the MG-specific antibody corresponding MG antigen, and investigated the candidate antigen genes and their recombinant proteins.In this study,2-week-old healthy meat chickens were artificially inoculated MG strain HS by nasal and then sera and respiratory tract rinse liquids were collected after 2 weeks of infection. Indirect ELISA, hemagglutination inhibition test and metabolism inhibition test were performed to evaluate the situation of MG-specific antibodies and further screened out the patent protective antigen of MG. The results were shown as following.1. MG specific IgY, IgA as major mucosal antibodies in respiratory tract washings (RTWs) from MG-HS infected chickensTested by indirect ELISAs after MG-infection, anti-MG IgA antibodies were detected at high level in RTWs (OD450=1.11±0.06,n=8) and at lower level in sera (OD450=0.46±0.09, n=8) (P<0.01) and anti-MG IgY titer were much higher in sera (OD450=2.47±0.17, n=8) and moderate in RTWs(OD450=0.72±0.08, n=8) (P<0.01); while anti-MG IgY or IgA in samples of healthy chickens were undetectedable (OD450<0.2, n=6). Our results suggested MG-infected chicken produced high titer of specific serum IgY antibodies, but also the IgY antibodies were largely secreted or transfered to the mucosal surface, which played a role together with IgA in mucosal immunity.2. MG hemagglutination inhibition antibodies (HI-IgY) in serum had no ability of inhibiting MG metabolismAnalyzed by hemagglutination inhibition test and metabolism inhibition test (MI), the mean HI-IgY of MG-infected (n=8) were at 1:64 in serum and 1:2 in RTWs, between which were significantly different (P<0.01)and the titer of MI antibodies were at 1.75±0.71(log2, n=8) in serum and 6.63±0.95 (log2, n=8) in RTWs, between which were significantly different (P<0.01). The results of all samples of serum and RTWs from healthy control were undetectable or negative. Our results indicated that HI-IgY unassociated with inhibition of MG metabolism.3. Anti-MG IgA from respiratory mucosa could inhibit MG growth and metabolismAs results shown above,2 weeks after infection, HI-IgY appeared largly in the serum and much less in RTWs; while metabolic inhibition antibody was detected mostly in RTWs but negatively in the serum. However, mucosal antibody IgA exist largely in RTWs and lower in serum, but the IgY antibodies were at high level in both serum and RTWs. Our results suggested (1) anti-MG IgY could also be used as a mucosal antibody and cooperate with IgA in mucosal immunity, (2) high titer serum hemagglutination inhibition antibody (targeting hemagglutinin) had no inhibition effect on MG metabolism and growth, (3) mucosal anti-MG IgA play a critical role in inhibiting growth and metabolism of MG, and the corresponding antigen may be the protective antigen candidate.4. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potent protective antigen of Mycoplasma gallisepticumWe used lysis of MG-HS as antigen, RTWs as primary antibody and goat anti-chicken IgA or IgY conjugated with horseradish peroxidase as second antibody in Western blot and analyzed the major bands by mass spectrometry and bioinformatics analysis. As results, two potent protective antigens were screened out, which included glyceraldehydes-3-phosphate dehydrogenase (GAPDH),37KD, a kind of sugar metabolism related enzymes and VlhA4.12 protein,75KD, as a member of a hemagglutinin family in MG, reported as pMGA previously.5. Expressing recombinant GAPDH and V1hA4.12 in Ecoli.Candidate antigen genes were cloned and among which TGAs encoding tryptophan were synonymously mutated as TGGs. The recombinant proteins of the mutant genes was expressed in Ecoli BL21(DE3) and identified by Western blot. As results, GAPDH or V1hA4.12 gene with mutation was expressed respectively at high level in E.coli, and the recombinant was reacted with RTWs of MG-infected chicken and showed strong immunologic activity. However, mouse anti-GAPDH IgG had no growth/metabolic inhibition of MG, which indicated anti-GAPDH IgY had no effect on MG metabolism.In conclusion, our study and results may expand a new insight of mucosal immune mechanism of MG and provide new potent candidate antigen for development of MG immune prevention and subunit vaccines.