Construction Of Sheep MBL Eukaryotic Expression Vector And Screening Of Disease Resistance Associated MiRNA

Sheep Mycoplasma pneumonia(MP) is a chronic respiratory diseases, whoes pathogenic mechanism is relatively complex and clinical diagnosis,prevention are difficult.The main characteristics of sheep Mycoplasma pneumonia is cough, breathing, progressive weight loss and chronic proliferative interstitial pneumonia. As a member of natural immune system, Mannan-binding lectin(MBL) mainly existing in the human and animal’s blood and liver, can specifically combine with bacteria, mycoplasma and other pathogenic microorganisms carbohydrate structures, direct or mediate by activation of the complement lectin pathway,play an important rolein the body’s natural immune defense, immune surveillance.Object: Analyze the role and the effect of MBL protein playing in innate immunity of Mycoplasma pneumoniae infection,to verify the relationship between MBL protein and sheep Mycoplasma pneumonia.Methods:(1)liver of healthy sheep were collected to extract RNA and RT to get cDNA.Primers were designed to amplify MBL coding region sequence.Interest gene MBL and the eukaryotic expression vector pTT5 were connected DNA ligase after double digested by EcoRI and Hind III to constructe sheep MBL gene eukaryotic expression plasmid pTT5-MBL.The pTT5-MBL were transiently transfected CHO cells and 293 cells to obtain CHO-MBLand 293-MBL which can express recombinant MBL proteins and to separate and purificate recombinant MBL protein.Methods to separate and purificate of MBL protein from serum of human were used to isolate and purificate of sheep plasma-derived protein MBL protein.sheep Mycoplasma pneumoniae were were artificially injected by intratracheal,Clinical symptoms were observed daily after artificial infection.Sheep of infected group were respectively injected plasma-derived MBL protein, recombinant MBL protein and saline after 14 d of injection and then observe clinical symptoms.Sheep were slaughtered at four weeks after protein injection.(2)Fresh blood and serums were collected respectively at one day before the protein treatment(-1d), one week(7d), two weeks(14d), three weeks(21d), four weeks(28d).ELISA and Quantitative PCR were used to analysis the concentration changes and the expression differences in serum of MBL, TNF-α, IFN-γ, C1,C3,IL-2 and IL-4.(3)Sick sheep of resistance groups and vulnerable groups of were slaughtered at four weeks after protein injection.Liver tissues were collected,stored in liquid nitrogen and then were sent to biotechnology companies to do mi RNA HiSeq send high-throughput sequencing.Sequencing results were analysed by bioinformatics analysis to screening of differentially expressed mi RNA.Results:(1) Sheep MBL gene eukaryotic expression vector pTT5-MBL was successfully constructed.Recombinant MBL who have MBL protein activity were successfully Expressed and purified.Plasma MBL protein were successfully isolated and purified,whose MBL protein activity.Artificial infection group after infection have appeared cough, loss of appetite, runny nose, diarrhea and other symptoms typical of mycoplasma.The results of sheep Mycoplasma pneumoniae recycling culture, biochemical identification and Mycoplasma Mycoplasma PCR identification showed that artificial infection was successful and artificial infection group were all successful infected in sheep Mycoplasma pneumoniae.After MBL protein injection, the injection protein group had different degrees the temperature drops,and the appetite recovery.Compared with the experimental control group, the symptom necropsy vision changes andpathological changes of the two group of the injected protein were slightly reduced.(2)After protein injection,the serum concentrations of IL-2,IL-4,IFN-γ,TNF-α and C3 declined first and ascended from 14 d after protein injection,and were higher than the experimental control group at different times,C1 ascended first and declined,this explanate that cellular and humoral immune function has been strengthened after protein injection.After protein injection,the expression levels of MBL, TNF-α, IFN-γ, C1,C3,IL-2 and IL-4 were increased in varying degrees, and then gradually decreased,this explanate that with the development of the course, the inflammation has been soothing.(3)Using high-throughput sequencing technologies selected multiple immune associated mi RNAs and signaling pathways.Conclusion:(1)A method of sheep MBL gene eukaryotic expression vector was constructed.(2)MBL protein immunotherapy can short-term relieve the clinical symptoms and pathological changes of sheep Mycoplasma pneumoniae,enhance cellular and humoral immunity of the body, improve the body’s resistance to disease, relieve inflammatory conditions.(3)Using high-throughput sequencing technologies selected multiple immune associated mi RNAs and signaling pathways.