Full-length CDNA Cloning, Molecular Characterization And Differ Ential Expression Analysis Of Interleukin1β And Interleukin1Receptor Antagonis From Ovis Aries

Interleukin1β (IL-1β) and interleukin1receptor antagonist (IL-1Ra) belong tothe interleukin1family (IL-1).IL-1β can coordinate other cytokines during theactivation process of T and B lymphocyte and play a role as a former inflammatorycytokines in promoting the synthesis of a variety of inflammatory substances. IL-1βand IL-1Ra can be combined with IL-1RI because of the similar structure. Thecombine of IL-1Ra with IL-1RI will not trigger signal transduction so that canadersely affect the biology function of IL-1β.Although IL-1α or IL-1β play animportant role in the resist process of the host to invasion of the pathogenicmicroorganisms, the amount of the expression of them will cause severe inflammationand autoimmune diseases.Keep the balance level of IL-1α or IL-1β and IL-1Ra play acrucial role to maintain the body steady and prevent diseases. Studies have shown thatmany diseases can cause specific expression patterns of IL-1β and IL-1Ra.It isexpected to provide new ideas to prevention, diagnosis and treatment of thework-related diseases by analyzing differentially expression patterns of IL-1β andIL-1Ra in disease.Brucellosis is a zoonosis caused by brucella, the clinical features of it containlong-term fever, sweating, joint pain and hepatosplenomegaly and so on. Bengal plateagglutination test (BPAT), as a kind of quick and reliable serological diagnosticmethods, is widely used in livestock of the quarantine of brucellosis. In livestockproduction, inoculating with brucella attenuated vaccine is an effective measures forprevention of brucellosis. Strong brucella strains infection and vaccination can both cause a positive BPAT result in livestock, and there are no effective measures forfurther identification of the source of infection. It leads to a situation that thevaccinated livestock is handled as a sick animals, and bring serious difficulty inbrucellosis purification.By estabishing the suppression subtractive hybridization (SSH)cDNA library of sheep leukocyte layer infected by Brucella virulent or attenuatedstrain2, differential expression genes between IL-1β and IL-1Ra was screened, and itis predicted that there may be specific differential expression pattern of IL-1β andIL-1Ra during the infected process of sheep by Brucella virulent or attenuated strain2.Revealing this differential expression pattern may provide a theoretical basis foridentification the source of infection of the BPAT+livestock, and is of greatsignificance in prevention, diagnosis and treatment of brucellosis.1. IL-1βand IL-1Ra full-length cDNA cloning, expression and the analysis ofmolecular characterizations from ovis ariesBased on the partial sequences of IL-1β and IL-1Ra obtained from the SSHcDNA library, full-length cDNA sequence of IL-1β and IL-1Ra were cloned throughnested PCR in use of RACE technology. The IL-1β is with a full-length of1494bpwhich concludes an open reading frame of801bp. It could encode266amino acidresidues and the molecular weight of IL-1β protein from Ovis aries is predicted to be30.6kDa.The IL-1Ra is with a full-length of1228bp which concludes an openreading frame of525bp. It could encode174amino acid residues and the molecularweight of IL-1Ra protein from Ovis aries is predicted to be19.8kDa. Using themolecular biological technique, the recombinant expression plasmid pET-30a-IL-1βand pET-28a-IL-1Ra about IL-1β and IL-1Ra from Ovis aries wereestablished.Recombinant expression protein orIL-1Ra and orIL-1Ra were obtainedafter inducing expression and purification by affinicty chromatography technology.The molecular biological information of IL-β and IL–1Ra was analysed thoughbioinformatics software and related website.The biological activity of orIL-1β wasmeasured by thymocyte proliferation assay. The biological activity of orIL-1Ra wasmeasured by detecting the antagonistic action from orIL-1Ra to the cell-killing effect on melanoma B16cells of IL-1β.2. Preparation of monoclonal antibody of IL-1βand IL-1Ra from ovis ariesMice were immunized with orIL-1β and orIL-1Ra. B cells from the immunizedmice were fused with SP2/0myeloma cells by cell fusion techniques. Two hybridomacell lines which could secrete anti-IL-1β antibody and anti-IL-1Ra antibody werescreened by indirect ELISA and Western Blot and the antibody subclasses were bothidentified to be IgG1. Two kinds of monoclonal antibodies were pruduced by induceascites method and purified by G protein affinity chromatography system.3. Space-time differentially expression characteristics analysis of IL-1βandIL-1Ra from Ovis ariesThe above-prepared monoclonal antibodies were used to detect the relativecontent of IL-1β and IL-1Ra in different tissues of healthy Ovis aries. Result showedthat it had the highest content of IL-1β in spleen and the lowest in leukocyte while thehighest content of IL-1Ra in muscle and the lowest in the lung.E. coli,Listeria, Salmonella, Brucella attenuated S2and Brucella inactivated S2were used to infect peripheral blood leukocytes from Ovis aries，the content of IL-1βand IL-1Ra in cell culture supernatant were measured in different time points afterinfection. The result showed that the content of IL-1β and IL-1Ra were all increasedgradually except the one infected by Brucella inactivated S2which was increased andthen decreased.Analysis the different transcription patterns of IL-1β and IL-1Ra of Ovis ariesinfected by Brucella virulent or attenuated strain2through fluorescence quantitativePCR.The expression quantity of IL-1β gene in total RNA in two groups both showed atrend of increase after the first fall, the group infected by Brucella virulent got to thelowest expression amount at the14st day and reached the highest quantity at the40thday after infected. The group infected by Brucella attenuated strain2got to the lowestexpression amount at the7th day and reached the highest quantity at the21st day afterinfected. The expression quantity of IL-1Ra gene in two groups both showed a trendof increase.Analysis the content of IL-1β and IL-1Ra in plasma of Ovis aries after infected by Brucella virulent or attenuated strain2by Western Blot.The content ofIL-1β in plasma after infected by Brucella virulent or attenuated strain2increasedwith the extension of infection time and then returned to normal levels. The increasedlevel of the group infected by Brucella virulent is higher than the group infected byBrucella attenuated strain2.The content of IL-1Ra in plasma after infected byBrucella virulent or attenuated strain2were both showed a significant increase at the40th day after infected, and the increased level of the group infected by Brucellavirulent is higher than the group infected by Brucella attenuated strain2.In this study, IL-1β and IL-1Ra from Ovis aries were studied as the researchobject, the molecular properties and differencial expression patterns of them underdifferent space-time conditions were analyzed based on the cloning of the full lengthcDNA sequence, the expression in genetic engineering and the preparation ofmonoclonal antibodies. This study provided new ideas to identification the source ofthe infection of BPAT+livestock.