| Mycoplasma hyopneumoniae can cause Mycoplasma pneumonia of swine(MPS).After entering into the respiratory tract of pigs,M.hyopneumoniae can cause respiratory system disorders,severe lung lessions,and reduce immune function.More and more evidence shows that after M.hyopneumoniae enters the body,the immune system cannot completely eliminate it,even if the use of antibacterials can only temporarily relieve symptoms.This has led to the long-term existence of M.hyopneumoniae in pigs,and it can cause continuous infection of the body which has brought great difficulty to humans to prevent and control the disease and caused significant losses to the pig industry worldwide.Autophagy is a highly conserved pathway to maintain homeostasis in cells.It is a process in which damaged organelles and misfolded proteins are encapsulated by vesicles of endoplasmic reticulum and Golgi membrane to form autophagosomes.Autophagosomes fuse with lysosomes to form autophagosomes and degrade their contents.At the same time,many pathogens can escape clearance of immune system through autophagy and survive in cells for a long time.Previous studies in our laboratory have shown that M.hyopneumoniae can enter lung tissue cells and induce autophagy,but it is not clear which kind of lung tissue cells M.hyopneumoniae can enter.Therefore,the purpose of this study is to determine the lung tissue cells that M.hyopneumoniae can enter,if it can enter the porcine alveolar macrophages,whether it can induce autophagy of porcine alveolar macrophages,and through which signaling pathways it can induce autophagy.(1)Preparation and application of Mhp366-N polyclonal antibodyObjective:To prepare the specific antibody of M.hyopneumoniae for the subsequent detection of M.hyopneumoniae.Method:The Mhp366-N protein was expressed by recombinant E.coli BL21(DE3)-p ET28a(+)-Mhp366-N,and the Mhp366-N protein was purified by Ni~+column and the concentration of Mhp366-N was adjusted to 2 mg/m L.The polyclonal antibody of Mhp366-N was produced by immuning mice.The titer of the polyclonal antibody was determined by ELISA,and the application of polyclonal antibody used in Western blot and immunofluorescence was tested.Result:The titer of Mhp366-N polyclonal antibody was up to 1:512 000.The best dilution of polyclonal antibody for Western blot was 1:100 000,and the dilution for immunofluorescence test was 1:1 000～1:1 000 000.Conclusion:Mhp366-N polyclonal antibody was prepared,and it can be used in Western blot and immunofluorescence assay.(2)M.hyopneumoniae induces autophagy in porcine alveolar macrophagesObjective:To determine whether M.hyopneumoniae can induce autophagy in infected porcine alveolar macrophages.Method:Porcine lung with typical gross lessions of MPS and without any gross lessions were collected to isolate porcine alveolar macrophages.The P36 gene of M.hyopneumoniae was detected by nested PCR,the number of autophagosomes was detected by transmission electron microscope,the expression of antophagy-related proteins was determined by Western blot,and the co-localization of M.hyopneumoniae and LC3 and the number of LC3dots were determined by immunofluorescence.After infection of 3D4/21 cells with M.hyopneumoniae AH strain,the number of autophagosomes,the expression of autophagy-related protein,the co-localization of Mhp366 and LC3 and the number of LC3 fluorescent puncta were also detected.Result:The isolated porcine alveolar macrophages were divided into M.hyopneumoniae positive and negative cells.Immunohistochemistry result showed that M.hyopneumoniae could enter into type I and type II alveolar cells,alveolar macrophages and bronchial epithelial cells.The number of autophagosomes,the expression of antophagy-related proteins LC3-Ⅱ,Beclin 1,Atg5 and Atg12-Atg5,yellow fluorescent puncta representing the co-localization of Mhp366 and LC3 in M.hyopneumoniae positive porcine alveolar macrophages were significantly more than those in M.hyopneumoniae negative porcine alveolar macrophages.The same results were obtained in 3D4/21 cells infected with AH strain.Conclusion:M.hyopneumoniae infection induced autophagy in porcine alveolar macrophages.(3)Research on the signaling pathway of autophagy induced by M.hyopneumoniaeObjective:To determine the signal pathway of autophagy induced by M.hyopneumoniae-infected cells.Method:Western blot was used to detect the expression of LC3-II and Beclin 1,as well as the phosphorylation of JNK,ERK,p38and Akt,after the infection of 3D4/21 cells by M.hyopneumoniae AH strain.3D4/21cells pretreated with JNK,ERK and Akt inhibitors were infected with AH strain.The expression of LC3-II and Beclin 1 was detected by Western blot.The number of LC3puncta and the co-localization of LC3 with Mhp366 in the cells were detected by indirect immunofluorescence.Result:The phosphorylation of JNK,ERK and Akt were increased,while the phosphorylation of p38 was almost unchanged in 3D4/21cells infected with AH strain.The expression of LC3-II and Beclin 1 and the number of LC3 fluorescent puncta were significantly decreased in JNK and Akt inhibitor treated 3D4/21 cells infected with M.hyopneumoniae,while the expression of LC3-II and Beclin 1 and the number of LC3 fluorescent puncta were not significantly changed in ERK phosphorylation inhibitor treated group.Conclusion:M.hyopneumoniae may induce autophagy through MAPK/JNK and PI3K/Akt signaling pathways. |
