布鲁氏菌Omp25蛋白抑制巨噬细胞分泌IL-12的分子机制研究

发布时间：2018-04-01 08:43

  本文选题：Omp25蛋白　切入点：重组腺病毒　出处：《西北农林科技大学》2015年硕士论文

【摘要】：布鲁氏菌病(Brucellosis)是由布鲁氏菌(Brucella)引起的一种人畜共患慢性传染病,具有传播范围广、感染动物种类多和危害大的特点。布鲁氏菌可经多种途径感染人和动物,对人类健康和畜牧业的发展造成严重危害。巨噬细胞在机体先天免疫中发挥着重要的作用,但是,布鲁氏菌不仅可以抵抗巨噬细胞的杀伤作用,而且可以在巨噬细胞中生存繁殖,并抑制巨噬细胞所介导的天然免疫反应。前期研究发现,布鲁氏菌外膜蛋白Omp25可以抑制巨噬细胞分泌TNF-α,提示Omp25在布鲁氏菌免疫逃逸特性中可能发挥着重要作用。然而,Omp25蛋白是否可以通过调控巨噬细胞IL-12的产生进而影响Th1细胞免疫功能还未见相关报道。本研究拟明确Omp25蛋白在调控巨噬细胞IL-12产生中的作用及调控机制,为进一步阐明布鲁氏菌免疫逃逸的分子机制奠定理论基础。研究工作取得如下结果:1.布鲁氏菌Omp25重组腺病毒的构建及鉴定从本实验室保存的p CI-neo-Omp25载体中PCR特异性扩增得到omp25基因(猪种布鲁氏菌S1330),将其插入经Xho I和Eco R V双酶切的p Shuttle-CMV载体中,基因序列测定显示omp25基因无突变,从而成功构建腺病毒穿梭载体p Shuttle-CMV-Omp25;Pme I线性化p Shuttle-CMV-Omp25和p Shuttle-CMV,通过电转化法使穿梭载体进入BJ5183-AD-1细菌中与骨架载体p Ad-Easy-1重组,重组腺病毒载体命名为p Ad-Omp25和p Ad-Blank;p Ad-Omp25和p Ad-Blank经Pac I线性化后转入293细胞中包装为重组腺病毒,命名为r Ad-Omp25和r Ad-Blank。r Ad-Omp25和r Ad-Blank感染PK15细胞、THP-1细胞和3D4/21细胞,RT-PCR和Western blot证明omp25在细胞内转录、Omp25蛋白在细胞内表达。2.Omp25对巨噬细胞分泌IL-12的影响及机制ELISA结果显示布鲁氏菌Omp25可以抑制LPS/R848诱导的THP-1分泌IL-12p70;Real-Time PCR结果显示Omp25可以降低LPS/R848诱导的THP-1细胞中IL-12B(p40)的m RNA水平,但是对IL-12A(p35)的m RNA水平没有显著的影响;Western Blot检测显示Omp25抑制LPS/R848诱导的THP-1中IκB的磷酸化;Real-Time PCR测定结果表明Omp25可以上调THP-1中的mi R-155、mi R-23b和mi R-21水平,而Real-Time PCR检测猪肺巨噬细胞3D4/21发现Omp25可以上调3D4/21中mi R-155和mi R-21,但是对mi R-23b没有显著影响。上述结果提示,布鲁氏菌Omp25可以显著抑制病原相关分子模式(如LPS、R848)诱导的单核巨噬细胞IL-12的产生,但是Omp25在布鲁氏菌感染人和猪巨噬细胞时发挥作用的方式可能不完全相同。综上所述,本研究成功构建了Omp25重组腺病毒,感染单核细胞THP-1,成功地表达了Omp25蛋白。实验证明Omp25蛋白抑制LPS/R848诱导的巨噬细胞产生IL-12。对其机制的研究表明Omp25上调THP-1中的mi R-155、mi R-23b和mi R-21,这些micro RNA可能参与调控Omp25对LPS/R848诱导的巨噬细胞IL-12产生的抑制作用;Omp25可以抑制LPS/R848诱导的THP-1中NF-κB信号途径,证明Omp25在转录水平抑制THP-1表达IL-12。
[Abstract]:Brucellosis (Brucellosis) by Brucella (Brucella) caused by a zoonotic and chronic infectious disease, with widespread infection animal variety and harm characteristics. Brucella can infect human and animal through a variety of ways, the development of human health and animal husbandry caused serious harm in the innate immune macrophage. Play an important role, but can not only resist the killing effect of Brucella in macrophages, and can survive in macrophages and inhibit reproduction, natural immune reaction mediated by macrophages. The preliminary study found that Brucella outer membrane protein Omp25 can inhibit macrophage TNF- secretion, suggesting that Omp25 in immune escape of Brucella may play an important role characteristics. However, whether the Omp25 protein can affect the immune function of Th1 cells by macrophage IL-12 production It has not been reported. This study intends to produce a clear role in the regulation of Omp25 protein in IL-12 macrophages and the regulation mechanism, which lays the theoretical foundation for further clarify the molecular mechanism of Brucella immune escape. The research results are as follows: 1. the construction of Brucella Omp25 recombinant adenovirus vector and identification of P CI-neo-Omp25 stored in our lab in PCR. The specific amplified omp25 gene (Brucella suis S1330), P Shuttle-CMV Xho I was inserted into the vector Eco and R V double enzyme digestion in determination showed that the omp25 gene mutation gene sequence, and from the successful construction of adenovirus shuttle vector p Shuttle-CMV-Omp25; Pme I P Shuttle-CMV-Omp25 and P Shuttle-CMV linearization, the shuttle vector enter the BJ5183-AD-1 bacteria and the recombinant Ad-Easy-1 backbone plasmid P by electroporation method, recombinant adenovirus vector named P Ad-Omp25 and P Ad-Blank; P A D-Omp25 and P Ad-Blank by Pac I after linearization into 293 cells for packaging of recombinant adenovirus, named as R Ad-Omp25 and R Ad-Blank.r Ad-Omp25 and R Ad-Blank infection of PK15 cells, THP-1 cells and 3D4/21 cells, RT-PCR and Western blot that omp25 in vivo transcription, expression of Omp25 protein in cells in the effect of.2.Omp25 on IL-12 secretion of macrophages the results showed that the ELISA and the mechanism of Brucella Omp25 can inhibit LPS/R848 induced secretion of THP-1 IL-12p70; Real-Time PCR showed that Omp25 can reduce the IL-12B of LPS/R848 in THP-1 cells induced by M RNA (P40) level of IL-12A (p35), but no significant effect of the m RNA Western Blot showed that the Omp25 level; inhibit LPS/R848 induced THP-1 I K B phosphorylation; the test results of the Real-Time PCR show that Omp25 can up regulate the expression of THP-1 in MI R-155, MI R-23b and MI R-21, and Real-Time PCR detection of swine 3D4/21 Omp25 found that pulmonary macrophages could increase 3D4/21 in MI R-155 and MI R-21, but had no significant effect on MI R-23b. The results suggest that Omp25 of Brucella can significantly inhibit pathogen associated molecular patterns (LPS, R848) induced by monocyte macrophage IL-12 production, but Omp25 infection in human and pig macrophages play a role of Brucella the way may not be exactly the same. In summary, this study successfully constructed Omp25 recombinant adenovirus infected monocytes THP-1 successfully expressed Omp25 protein. The results indicated that Omp25 protein inhibited LPS/R848 induced macrophage to produce research on the mechanism of IL-12. showed that Omp25 up-regulated the expression of THP-1 in MI R-155, MI R-23b and MI R-21, the micro RNA may be involved in the regulation of Omp25 on inhibition of LPS/R848 induced macrophage IL-12; Omp25 can inhibit LPS/R848 induced THP-1 NF- K B channel It is proved that Omp25 inhibits the expression of IL-12. at the transcriptional level of THP-1

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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