Establishment And Application Of Real-time PCR Methods Of Interleukin 1β And Interleukin 1 Receptor Antagonist From Sheep(Ovis Aries)

Interleukin1β(IL-1β) and interleukin 1 receptor antagonist(IL-1Ra) are two major members of the interleukin 1 family. IL-1 family are not only an important part of the innate immune system, but also play a regulatory role on the acquired immune system. IL-1β can coordinate all kinds of cytokines to activate T, B lymphocyte differentiation. As the three-dimensional structure of IL-1Ra is similar to IL-1β,IL-1Ra can competitively bind to IL-1RI,but they can not form dimers and conduct signaling, thus IL-1Ra can antagonize the biological activity of IL-1β. A large of studies have shown that the balance between IL-1β and IL-1Ra is related to some diseases, such as arthritis, inflammatory bowel disease, granulomas and central nervous system diseases. Meanwhile, Our previous studies had found that there was a differential expression of IL-1β and IL-1Ra after sheep(ovis aries) were infected with bacteria such as Brucella.To establish reverse transcription real-time fluorescent quantitative PCR method of IL-1β and IL-1Ra from sheep(ovis aries), according to the Oa IL-1β(Gen Bank accession number KC425612) and Oa IL-1Ra(Gen Bank accession number KC425613)m RNA sequences, specific primers were designed to amplify the target gene. The two kinds of target genes were cloned into the p MD-18 T vector to obtain recombinant plasmids, and the two positive recombinant plasmids were respectively established standard curve, and performed sensitivity, repeatability and specificity analysis experiments. By melting curve analysis, there was a single peak and good repeatability. We got the regression equation, the correlation coefficient and amplification efficiency of IL-1 β: y =-3.463 x + 39.957, R2 =0.9999, Eff= 94.4%,and the coefficient of variation ranged from percent 0.28 to 1.45, the linear range was from 4.68 to 1.58 ×107 copies/μL, the lowest detection line was 4.68 copies/μL, the recovery of samples was percent 91.4. However,we got the regression equation, the correlation coefficient and amplification efficiency of the IL-1Ra : y =- 3.3888 x +41.452,R2 =0.9995, Eff=97.3 %, and the coefficient of variation ranged from percent0.08 to 1.23, the linear range was from 5.31 to 2.27 ×107 copies/μL, the lowest detection line was 5.31 copies/μL, and the recovery of samples was percent 88.3.Applying to the reverse transcription real-time quantitative PCR method in this experiment, 182 clinical samples related to sheep peripheral white blood cell infected with Brucella were analyzed the gene transcription abundance of IL-1β and IL-1Ra.The balance between IL-1β and the IL-1Ra was initially identified in transcription level. IL-1β and IL-1Ra gene transcription and quantitative detection method in clinical samples were established in this study, the discussion for the balance between IL-1β and IL-1Ra may have a further research as reference in assisting diagnosis of Brucellosis, maintaining homeostasis and treating diseases, and provide a scientific basis and quantitative monitor tool for subsequent clinical application of the method.