The Immune Regulation Effects Of Recombinant SPLUNC1 Protein In Argali Hybrid Sheep Infected With Mycoplasma Ovipneumoniae

Objective: In this study,the Bashibai sheep(BS)and Argali hybrid sheep(AHS)were infected with Mycoplasma ovipneumoniae(MO)as disease model.To research the immunity and treatment effect of the recombinant short palate,lung and nasal epithelium clone 1 protein(r SPLUNC1)in ASH infected with MO,the ASH was treated with BS and AHS recombinant SPLUNC1 protein,then test the expression level of SPLUNC1 m RNA by real-time q PCR,and the IL-5?IL-6?IL-8?IL-9?IL-12 and IL-13 cytokines standard by ELISA.Methods:(1)Expression and Purification of r SPLUNC1: Culture the BS and AHS recombinant Pichia GS115/p PIC9K-SPLUNC1,collect the expression of 96 h,using Phenyl sepharose FF affinity chromatography,and SDS-PAGE method to detect the effect of expression and purification.(2)The immunologic effect of r SPLUNC1 on the BHS after infected with MO: Six BS are group A,eighteen AHS are random group B,C and D.All the sheep artificially infected with MO.After two weeks,detect the MO-antibody level.From the 5th day post-infection(DPI),group C and D treated with r SPLUNC1 protein,150?g/sheep.Group A and B injected with placebo.Collecting blood and separating serum at 0 d,2 d,5 d,7 d,14 d,21 d after infection,respectively.The level of IL-5,IL-6,IL-8,IL-9,IL-12 and IL-13 of serum were test by ELISA to compare cytokine concentration changes in all groups.(3)SPLUNC1 m RNA level change in AHS with injecting r SPLUNC1: Collect the oral pharynx and larynx of palate tissue before infection(0 d),after infection and treatment.And expression level of SPLUNC1 m RNA detected by real-time q PCR.(4)Therapeutic effect of r SPLUNC1 on AHS infected with MO: Group C and D treated with r SPLUNC1 protein until 21 DPI.At the end of the experiment,all the sheep were dissected to observe lung pathological changes and to conduct pathological grading.Results:(1)On the basis of the constructed BS and AHS recombinant Pichia GS115/p PIC9K-SPLUNC1 positive clones on methanol-induced expression of 96 h supernatant was collected,SDS-PAGE analysis showed BS protein molecular size 25.53 k Da purposes strip and AHS protein molecular size 25.96 k Da purposes strip.(2)All the serums were detected by MO antibody ELISA kit and the results were positive.On the 14 DPI to 21 DPI,the IL-5 level of group A highly significant lower than group B(P<0.01;P<0.01),and significantly lower than group C and D(P<0.05;P<0.05);on the 21 DPI,the IL-5 level of group C and D highly significant lower than group B(P<0.01).The level of IL-6 were increased,from the 14 DPI to 21 DPI,group C and D highly significant lower than group B(P<0.01;P<0.01),and group A highly significant lower than group B(P<0.01;P<0.01).The level of IL-8 were increased,on the 14 to 21 DPI,group C and D significantly lower than group B(P<0.05;P<0.01),group A highly significant lower than group B(P<0.0 1;P<0.01).On the 7 DPI,the group B IL-9 concentration was highly significant higher than group A,C,D(P<0.01).From the 14 DPI to 21 DPI,group C and D significantly lower than group B(P<0.05;P<0.01),and group A highly significant lower than group B(P<0.01;P<0.01).From the 7 DPI,the IL-12 in group A significantly lower than group B(P<0.05);on 14 DPI,group C and D lower than group B(P<0.05;P<0.01).On 21 DPI,group C and D highly significant lower than group B(P<0.01).On the 5 DPI,the concentration of IL-13 in group A significantly lower than group B?C and D(P<0.05).On the 7 DPI,group B significantly higher than group A,C and D(P<0.05;P<0.01).On the 14 DPI to 21 DPI,group C and D highly significant lower than group B(P<0.01;P<0.01).On the 21 DPI,group A higher than group C and D(P<0.05),and lower than group B(P<0.05).The IL-5,IL-9,IL-12 and IL-13 levels have significant changes after inject r SPLUNC1 protein.It suggested that r SPLUNC1 effects the immune of AHS after infected with MO.(3)On the 21 DPI,the SPLUNC1 m RNA expression of group C,D significantly higher than group B(P<0.01),and group A significantly higher than group B(P<0.01).(4)Transiently high temperature(40-41 ?),anorexia,haemorrhage in lung of group A.Group B with long high temperature(42-42 ?),serious moist sounds,diarrhea,cough and tubercle and liver-like changes in lung,even two of them dead.Slightly high temperature and cough,and haemorrhage and ecchymoses in lung of group C.Serous nasal discharge and cough,ecchymoses in lung of group D.Histopathological scores results: Scores of the group A,B,C and D were 8.8,18.7,13.2,14,respectively.The score of group C and D was significantly lower than group B(P<0.05),the score of group A highly significant lower than group B(P<0.01).It suggested that r SPLUNC1 treatment on AHS after infected with MO.Conclusions: Expresed the BS and AHS recombinant SPLUNC1.The SDS-PAGE results shows the single band sized 25.53 k Da and 25.96 k Da that purified by Phenyl Sepharose FF.Group C and D injected with BS and AHS recombinant SPLUNC1 protein.The concentration of IL-5,IL-6,IL-8,IL-9,IL-12 and IL-13 compared with the B group had a significant changed,indicating two kinds of recombinant SPLUNC1 protein regulates the argali hybrid sheep related cytokines after MO infection.Each level of SPLUNC1 m RNA result showed that the recombinant SPLUNC1 protein has a certain effect on AHS endogenous SPLUNC1 m RNA level.Autopsy and pathological score result showed that two kinds of recombinant SPLUNC1 protein on MO infected AHS has certain treament.