Preparation Of Antibody Against Chicken Macrophage Migration Inhibitory Factor(MIF) And The Mechanism Of MIF In Pulmonary Hypertension

[Purpose] In the species of human and experimental animals(mice and rats),macrophage migration inhibitory factor(MIF)was a multi-effect molecule which can act as cytokines,neuroendocrine hormones and enzyme properties.Till now,there were relatively few studies on MIF in poultry.In this study,rabbit anti-chicken MIF polyclonal antibody was prepared,and detected its application in inflammation model to obtain specific antibody,which would lay the foundation for the study of the biological function of MIF in poultry.This study also detected the expression of MIF in broiler pulmonary hypertension(PH),and the expression of related proteins and genes in the downstream signal pathway of MIF,to explore the mechanism of MIF participating in PH,and to provide theoretical basis to find new therapeutic targets for broiler PH.[Method] 1.P-EASY-E1 was used as a vector to express MIF in prokaryotic cells,and the recombinant MIF protein was purified by Ni-column affinity chromatography.New Zealand rabbits were used as a host to prepare MIF polyclonal antibodies.ELISA was used to detect antibody titer,and immunoblotting to detect antibody specificity.2.Used this antibody to detect the basic expression of MIF protein in the heart,liver,spleen,lung,kidney,thymus,bursa,and cecum of broilers by immunoblotting.3.Detected the expression of MIF protein in the heart,liver,spleen,lung,and kidney of broilers in LPS induced inflammation models by immunohistochemistry and immunoblotting.4.4-5 weeks broilers with clinical PH were collected as experimental(PH)group,while the healthy broilers were taken as control group.The protein expressions and distributions of MIF,Ras,p-Raf,p-MEK,p-ERK,p-Akt,p-GSK3β,cyclin D were detected by immunoblotting and immunohistochemistry.The related m RNA expression levels of MIF,Raf,ERK,Akt,GSK MIF,Raf,ERK,Akt,GSK3β,cyclin D,Rb,E2 F were determined by q RT-PCR.[Result] 1.The prokaryotic expression system of chicken MIF,pEASY-E1-ChMIF was successfully constructed.The expression condition was that the recombinant MIF protein was soluble when induced by 0.05 m M IPTG at 28℃ for 5h.The Ni-NTA column was used for purification.The purified recombinant chicken MIF protein was obtained when the imidazole solution was 200 m M,SDS-PAGE showed that almost no mixed proteins existed.The titer of the antibody was 1:102400,which showed high sensitivity and specificity.A highly specific polyclonal antibody against chicken MIF was obtained with a titer of 1:102400.2.The Western blotting results showed that MIF protein had basal expression in the heart,liver,spleen,lung,kidney,thymus,bursa,and cecum of chicken,with high expression in the kidney and thymus,low basal expression in the lung and heart.3.In the inflammatory model,the expression of MIF in the heart,liver,spleen and lung of the chicken was significantly higher than that of the control group,which participated in the inflammatory reaction,while no significant change of MIF expression was detected in kidney compared with the control.4.IHC and WB results showed that the expression levels of MIF,p-Raf,p-ERK,p-Akt,p-GSK3β,cyclin D1,p-Rb,E2 F proteins in PH group were significantly higher than that of the control.The results showed that,the m RNA expression of MIF,Raf,ERK,Akt,GSK3β,cyclin D1,Rb,E2 F in PH group were significant higher than that of control.[Conclusion] 1.The rabbit anti chicken MIF polyclonal antibody was obtained,which has strong specificity for chicken.The polyclonal antibody can be used for immunohistochemical and Western blotting detection of chicken MIF protein.2.The basal expression of chicken MIF is fundamentally different in different tissues,which may imply different levels of involvement in immune responses.3.The MIF might be involved in the inflammatory response of broilers,and MIF content in the lung,heart,liver and spleen could be a new target for the treatment of broiler inflammatory diseases in the future.4.MIF participated in the occurrence of PH,possibly through Raf/ERK and Akt/GSK3β pathways by increasing the expression of cyclin D1,phosphorylation of Rb,and transcription function of E2 F,thereby induced cell proliferation,caused pulmonary artery vascular remodeling,and lead to pulmonary hypertension.