The Mechanism Of PBMC Apoptosis Induced By Mycoplasma Gallisepticum GroEL Protein And Its Value In Diagnostic

Mycoplasma gallisepticum(MG)is the pathogen that cause chronic respiratory diseases in chickens.The clinlcal symptoms of chickens infected by MG are cough,rhinitis,sneezing and tracheal rale.In addition,chickens infected with MG are susceptibility to other pathogenic and are easy to cause secondary infection leading severely respiratory syndrome,which causes enormous economic losses in poultry industry.Till now,the mechanism of MG pathogenic is still unknow and the level of prevention and control technology is relatively weak.In poultry farms,the infection rate of MG is as high as 70%~80%.Therefore,it is of great significance to research on the pathogenesis of MG and explore its effective immunogen protein.Mycoplasma lacks cell wall,and the cell membrane is the main structure that MG contact with the outside world.And many lipid-associated membrane proteins(LAMPs)on the surface of the membrane.The GroEL protein is one of the components of LAMPs of MG and GroEL can induce apoptosis of DF-1 cells,which was discovered by previous studies in our lab.However,DF-1 cells are passaged cell lines,which could not truly reveal the damage occurred in the immune system of chicken.In this study,peripheral blood monouclear cells(PBMC)were isolated from chicken peripheral blood,and was used to explore the apoptosis of PBMC induced by GroEL protein.Firstly,the recombinant expression plasmid of GroEL was constructed,and then rGroEL protein was expressed and purified.Flow cytometry and western blot were used to detect the apoptosis of PBMC induced by rGroEL protein.The results showed that the apoptotic rate of PBMC increased with the dose of rGroEL protein as well as the expression of caspase 3/8/9.These demonstrate that rGroEL protein can induce apoptosis of PBMC.To further study the mechanism of apoptosis induced by rGroEL Protein,Annexin A2 which can interact with GroEL protein was preliminarily screened in our laboratory.In this study,in order to confirm whether the apoptosis of PBMC induced by rGroEL protein was related to Annexin A2,the rGroEL protein was added into culture medium containing PBMC.Laser confocal microscopy was used to observe PBMC labeled with fluorescent antibodies after adding anti-GroEL and anti-Annexin A2 monoclonal antibodies respectively.The results showed that rGroEL protein and endogenous Annexin A2 can co-localize on the surface of PBMC membrane.Western blot analysis showed that the expression of Annexin A2 protein increased after PBMC stimulated by GroEL protein;Using rGroEL protein stimulates PBMC whoes mRNA of Annexin A2 interfered by siRNA,the results of flow cytometry analysis showed that the apoptotic rate of PBMC decreased significantly after the expression of Annexin A2 interferenced.The above results demonstrated that MG GroEL protein can induce host cell apoptosis by interacting with Annexin A2.Because of the important role of GroEL protein and its role as MG immunogen,In this study,an indirect ELISA method was established using rGroEL protein as antigen to test whether GroEL protein can be used as diagnostic antigen.Results showed that the indirect ELISA method has well sensitivity and no cross-reactivity with the positive sera of Mycoplasma Synoviae(MS).However,the coincidence rate of indirect ELISA method with commercialized detection kit for the same batch of samples was only 63.34 %.Therefore,rGroEL protein used as a diagnostic antigen for MG needs further verification.