Study On The Relation Between Macrophage Migration Inhibitory Factor And H5N1 Highly Pathogenic Avian Influenza Viral Pneumonia

The frequent outbreak of highly pathogenic avian influenza has brought a serious disaster to many poultry cultivation in China and even in the world.Avian influenza virus(AIV) has spanned the avian to infect the mammalians.In recent years, AIV has broken through the species barrier to contract human beings,which is serious threatening the health of people. The scope of host infected AIV is being enlarged ,mainly including bird, duck,tiger,lion,humanbeing.H5N1 AIV has posed a strong pathogenicity to tiger.The tigers infected with H5N1 AIV mainly display the pathology of pneumonia,which still has very high mortality.It has caused great economic loss to the zoo and the country.Many results of research have suggested that the pathogenicity of highly pathogenic AIV be related to the cytokines storm.The important agent of the pathogenicity may be the disequilibrium of the cytokines induced by the H5N1 AIV. We has not completely comprehended the pathogenesis of H5N1 AIV, although the study on the cytokines storm could supply a certain clue.The data about the change circumstance of cytokines which released by the organism infected by H5N1 AIV is limited ,especially about the model of animals.It is difficult to illuminate which kinds of the cytokines are closely related to the pathogenicity.ICAM-1 and its receptor have been claritied to play an important role in the inflammation caused by highly pathogenic avian influenza.The purpose of this study is to determine the dynamic levels of MIF in the lung and serum of the mouse which challenged with H5N1 AIV isolated from tiger.The study will be contributed to understand the pathogenic mechanism of H5N1 AIV and will provide the target of the research on remedy medicine.The recombinant plasmid expressing the MIF protein of the mouse was constructed to express the MIF protein of the mouse.The recombinant protein was demonstrated to acquire the high performance and have perfect reactionogenicity by SDS-PAGE,the gel scanning,Western blot.The MIF protein was purification by Ni2+ HisTrap affinity columns.So the material fundament was created to detect the MIF in the serum of mouse by ELISA method.The method of indirect competitive BA-ELISA was constructed by utilizing the purified MIF protein and many referred agents were optimized.The regression equation of the MIF standard preparation was y=38.288x-90.271,the square vaule of R was 0.9931.The curve had displayed good linear correlation when the concentration of the competitive antigen ranged from0.5ng/mL to 16ng/mL.The expression levels of MIF protein in the serum of the mouse were determined by the conducted BA-ELISA method.The results showed that the expression levels of MIF protein were reached a peak at 12hrs post infection with H5N1 virus isolated from tiger.It also verified the role of MIF in the early phase of inflammation. After 30h,60h,84h challenged withH5N1 AIV, the expression levels of MIF protein were also higher than control mice.This result suggested that MIF participate in the course of the highly pathogenic avian influenza viral pneumonia.In order to detect the MIF mRNA in the lung of mouse,we developed the real-time RT-PCR.The standard preparation ofβ–actin and MIF plasmid were constructed to apply the real-time fluorescence quantitative RT-PCR based SYBR Green I.The cycle numbers,the anneling temperature and the primer final concentration were optimized to determine the minimum primer concentrations giving the lowest threshold cycle and the maximum signal-noise fluorescence ratio while minimising non-specific amplification The equation of the dynamics curve ofβ–actin and MIF wereβ–actin:Ct=-2.946293×LogCopynumber+41.117092;MIF:Ct=-3.140122×LogCopynumber+43.762676.The ineal detection scope was ranged from 103 to108copies/reaction.The perfect reproducibility was displayed by the results of the intraassay and interassay variation.The expression levels ofβ–actin and MIF mRNA in the lung were determined by the optimal real-time fluorescence quantitative RT-PCR based SYBR Green I reaction.The results had showed that the relative expression levels of MIF mRNA were in the peak phase after 6h challenged with H5N1 AIV isolated from tiger.After 24h,48h,72h challenged by H5N1 AIV, the relative expression levels of MIF mRNA were also higher than other hours.The results indicated that MIF was a pivotal inflammation mediator in the occurance and the development of the highly pathogenic avian influenza viral pneumonia . This may be referred to the excessive activation of macrophage. The results of the study have implied us that MIF may be participate in the forming of the cytokines storms in the early phase infected with H5N1 AIV as a critical one.We primarily developed the theory basis in the purpose of revealing the pathogenesis of the highly pathogenic avian influenza viral pneumonia,also created unique ideas to the research of medicines which applied to prevent and treat the H5N1 influenza.