The Immunomodulatory On Effect Of Recombinant Ubiquitin ISG15Protein In Infected Mycoplasma Ovine Pneumoniae In Argali Hybrid Sheep

Objective:In this study, the research objects were the Argali hybrid sheep and Bashibai sheep,the former was Mycoplasma ovipneumoniae susceptible,while the latter was not,to study the effect of ISG15protein of Argali hybrid sheep and Bashibai sheep, while artificially infected Mycoplasma ovipneumoniae,used the real-time PCR and ELISA method to analysis the various cytokines changes from the level of the protein and gene expression, studied the role of ISG15protein in the congenital immune regulation and therapeutic.Method:(1)Proliferation culture of the Mycoplasma ovipneumoniae,measured the bacterial concentration and diluted to the artificial infection concentration,then the18Argali hybrid sheep were randomly divided into three groups,group A,B,C,group D was6Bashibai sheep, then injected the Mycoplasma ovipneumoniae into the intratracheal, everyday observed the clinical symptom and detected the rectal temperature, three weeks later, randomly collected the nasal swab of nasal secretion from the test sheep, recycling cultured Mycoplasma ovipneumoniae, and the biochemical identification was to detect whether the Mycoplasma ovipneumoniae caused the sheep sick.(2)Used the purified protein to treat the sheep that infected Mycoplasma ovipneumoniae in test one, group A was susceptible Mycoplasma ovipneumoniae sheep,daily injected with saline;group D was not susceptible Mycoplasma ovipneumoniae sheep, daily injected with saline;group B was treated with recombinant ISG15protein of Argali hybrid sheep,each one with200μ g everyday, observed the clinical symptom and detected rectal temperature; anatomically observed the lung pathological changes of the dead sheep, compared the differences between the test sheep after treatment.(3)During the process of infection test and treatment test, respectively,the day before challenged, the day1,7,14and21after challenged, collected fresh anticoagulant, separated the lymphocytes. Finally,the expression levels of ISG15, IFN-γ, IL-12were detected by real-time PCR,and compared the expression levels of cytokine among the test sheep.(4)Because the real-time PCR method failed to detect the cytokines IL-2, IL-4, IL-6,therefore,during the process of infection test and treatment test, respectively,the day before challenged, the day1,7,14and21after challenged, collected fresh blood, separated the lymphocytes. Finally,the expression levels of IL-2, IL-4, IL-6were detected by real-time PCR,and compared the expression levels of cytokine among the test sheep.Results:(1)Mycoplasma special medium turned yellow that indicated the mycoplasma isolated and cultured was successful; artificial infected entire flock was successful, all sheep showed varying degrees of fever, cough, loss of appetite, diarrhea and other symptoms after infection. Three to four weeks after challenged,group A had dead sheep,the mortality rate was50%, the necropsy of the lung surface found serious change of meat, liver degeneration, hemorrhage points and bleeding spots.There were no dead sheep among group B,C and D,but some sheep of group B and C became cough and diarrhea,autopsy after the test, lung had bleeding and bleeding spots.However, group D only became a transient increase in body temperature after challenging, then returned to normal, necropsy lung surface, there was slight bleeding point after the test. There were no dead sheep among the reatment group and Bashibai of sheep that were not susceptible group, and the lesions of Bashibai sheep was lighter than the treatment group.(2) In the first test day, the expression level of IFN-y was slight decline in the test sheep compared with before challenged,the day14, group B was significantly higher than group A and D (P<0.05),the day21, group B,C were significantly higher than group A and D (P<0.05),treatment increased the expression level of IFN-y;From the day14to21, the expression level of ISG15was detected,group B,C and D were significantly higher than group A(P<0.05), ISG15expression levels of group B and C were significantly increased than non-treatment group A after treatment;At the day14and21, the IL-12expression level of group A was significantly higher than other groups(P<0.05).(3) The test sheep expressed IL-12at the same levels before challenged,group D was significantly lower than group A,B and C after challenged5th day (P<0.05); Compared the day14with the day7after challenged, group A gradually increased, group D gradually decreased, and there was no significantly change between group B and C,the IL-12expression levels of group B and C were stable compared with group A and D. From the day7to21after challenged, the IL-4expression levels of group B and C were significantly higher than group A and D(P<0.05), which indicated that IL-4expression levels of group B and C were maintained at a high level after treatment, there was no significantly change in group D; And the IL-6expression levels of group B, C and D were significantly increased(P<0.05),then remained at a high level, which indicated that group A had a higher degree of inflammation.Conclusion:Treated the susceptible Argali hybrid sheep with recombinant ISG15protein, found that the mortality and expression levels of immune factors of susceptible Argali hybrid sheep were significant different from the susceptible Argali hybrid sheep control group, the result showed that ISG15protein had immunomodulatory and therapeutic effects, it laid a new theoretical basis for the clinical treatment of Mycoplasma ovipneumoniae and the development of new drug research.