The Molecular Mechanism Of Inhibitory Effects Of Brucella Omp25 Protein On IL-12 Secretion In Macrophage

Brucellosis, a zoonotic disease caused by Brucella, is widely popular in the world and endangers the public health and safety. Brucella can invade the body via a variety of ways, so it poses a serious threat to livestock and human health. Macrophages play an important role in innate immunity of host, and they are the major cells in which Brucella proliferation in the body. Brucella inhibits the innate immune response which macrophages mediated. We have demonstrated that Brucella Omp25 protein had the inhibitory effects on TNF-α secretion of macrophage, suggesting Omp25 protein might be play a significant role in the characteristics of immune escape in Brucella infection. However, it has not confirmed whether Omp25 protein could regulate the secretion of IL-12 in macrophages and further inhibit the differentiation of Th1 cells and Th1 immune response. This study are aimed to clarify the effects and molecular mechanism of Brucella Omp25 protein on IL-12 secretion of macrophages, which will provide new insight into underlying the molecular mechanism of Brucella infection induced immune escape in vivo.The following results were obtained in the present study:1. Construction and identification of recombinant adenovirus Brucella Omp25The optimization of the omp25(B.suis 1330) gene was amplified by PCR from p CI-neo-Omp25 saved in our lab, then the omp25 gene was inserted into the vector p Shuttle-CMV by double digestion of Xho I and Eco R V, and constructed the adenoviral shuttle vector p Shuttle-CMV-Omp25 by genome sequence determination. The linearized shuttle vector p Shuttle-CMV-Omp25 and p Shuttle-CMV were transfected into BJ5183-AD-1 by electric transformation to recombination with p Ad-Easy-1, then amplifying and extracting the recombinant vector named p Ad-Omp25 and p Ad-Blank. We obtained the recombinant adenovirus named r Ad-Omp25 and r Ad-Blank by transfering the linearized p Ad-Omp25 and p Ad-Blank using Pac I to 293 cells. The m RNA of r Ad-Omp25 was identified by reverse transcription PCR and the Omp25 protein was identificated by Western blot after r Ad-Omp25 and r Ad-Blank infected PK15, THP-1 and 3D4/21.2. The effects and the mechanism of Omp25 protein on the secretion of IL-12 in macrophages ELISA and Real-Time PCR results showed that Omp25 protein suppressed the expression of IL-12 induced by LPS/R848 in protein levels and m RNA in THP-1 cells. Western blot showed that the phosphorylation of IκB, a key transcriptional regulatory factor of IL-12, was inhibited in LPS/R848-induced THP-1 cells. The Real-Time PCR detection showed that mi R-155, mi R-23 b and mi R-21 were up-regulated by the expression of r Ad-Omp25 in THP-1 cells.This study successfully constructed the recombinant adenovirus of Omp25 which successfully expressed Omp25 protein in PK15, THP-1 and 3D4/21. The results indicated Omp25 protein inhibited the secretion of IL-12 induced by LPS/R848 in macrophages. Further results showed that NF-κB transcriptional activity was suppressed by Omp25 protein in LPS/R848-stimulated THP-1 cells and the secretion of IL-12 induced by LPS/R848 in THP-1 was also decrease in transcription level. In addition, mi R-155, mi R-23 b and mi R-21 were un-regulated in Omp25-expressed THP-1 cell, suggesting that these micro RNAs might be involved in the regulatory roles of Omp25 in IL-12 expression in macrophage.