本文選題：Omp25蛋白　切入點(diǎn)：重組腺病毒　出處：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：布魯氏菌病(Brucellosis)是由布魯氏菌(Brucella)引起的一種人畜共患慢性傳染病,具有傳播范圍廣、感染動物種類多和危害大的特點(diǎn)。布魯氏菌可經(jīng)多種途徑感染人和動物,對人類健康和畜牧業(yè)的發(fā)展造成嚴(yán)重危害。巨噬細(xì)胞在機(jī)體先天免疫中發(fā)揮著重要的作用,但是,布魯氏菌不僅可以抵抗巨噬細(xì)胞的殺傷作用,而且可以在巨噬細(xì)胞中生存繁殖,并抑制巨噬細(xì)胞所介導(dǎo)的天然免疫反應(yīng)。前期研究發(fā)現(xiàn),布魯氏菌外膜蛋白Omp25可以抑制巨噬細(xì)胞分泌TNF-α,提示Omp25在布魯氏菌免疫逃逸特性中可能發(fā)揮著重要作用。然而,Omp25蛋白是否可以通過調(diào)控巨噬細(xì)胞IL-12的產(chǎn)生進(jìn)而影響Th1細(xì)胞免疫功能還未見相關(guān)報(bào)道。本研究擬明確Omp25蛋白在調(diào)控巨噬細(xì)胞IL-12產(chǎn)生中的作用及調(diào)控機(jī)制,為進(jìn)一步闡明布魯氏菌免疫逃逸的分子機(jī)制奠定理論基礎(chǔ)。研究工作取得如下結(jié)果:1.布魯氏菌Omp25重組腺病毒的構(gòu)建及鑒定從本實(shí)驗(yàn)室保存的p CI-neo-Omp25載體中PCR特異性擴(kuò)增得到omp25基因(豬種布魯氏菌S1330),將其插入經(jīng)Xho I和Eco R V雙酶切的p Shuttle-CMV載體中,基因序列測定顯示omp25基因無突變,從而成功構(gòu)建腺病毒穿梭載體p Shuttle-CMV-Omp25;Pme I線性化p Shuttle-CMV-Omp25和p Shuttle-CMV,通過電轉(zhuǎn)化法使穿梭載體進(jìn)入BJ5183-AD-1細(xì)菌中與骨架載體p Ad-Easy-1重組,重組腺病毒載體命名為p Ad-Omp25和p Ad-Blank;p Ad-Omp25和p Ad-Blank經(jīng)Pac I線性化后轉(zhuǎn)入293細(xì)胞中包裝為重組腺病毒,命名為r Ad-Omp25和r Ad-Blank。r Ad-Omp25和r Ad-Blank感染PK15細(xì)胞、THP-1細(xì)胞和3D4/21細(xì)胞,RT-PCR和Western blot證明omp25在細(xì)胞內(nèi)轉(zhuǎn)錄、Omp25蛋白在細(xì)胞內(nèi)表達(dá)。2.Omp25對巨噬細(xì)胞分泌IL-12的影響及機(jī)制ELISA結(jié)果顯示布魯氏菌Omp25可以抑制LPS/R848誘導(dǎo)的THP-1分泌IL-12p70;Real-Time PCR結(jié)果顯示Omp25可以降低LPS/R848誘導(dǎo)的THP-1細(xì)胞中IL-12B(p40)的m RNA水平,但是對IL-12A(p35)的m RNA水平?jīng)]有顯著的影響;Western Blot檢測顯示Omp25抑制LPS/R848誘導(dǎo)的THP-1中IκB的磷酸化;Real-Time PCR測定結(jié)果表明Omp25可以上調(diào)THP-1中的mi R-155、mi R-23b和mi R-21水平,而Real-Time PCR檢測豬肺巨噬細(xì)胞3D4/21發(fā)現(xiàn)Omp25可以上調(diào)3D4/21中mi R-155和mi R-21,但是對mi R-23b沒有顯著影響。上述結(jié)果提示,布魯氏菌Omp25可以顯著抑制病原相關(guān)分子模式(如LPS、R848)誘導(dǎo)的單核巨噬細(xì)胞IL-12的產(chǎn)生,但是Omp25在布魯氏菌感染人和豬巨噬細(xì)胞時(shí)發(fā)揮作用的方式可能不完全相同。綜上所述,本研究成功構(gòu)建了Omp25重組腺病毒,感染單核細(xì)胞THP-1,成功地表達(dá)了Omp25蛋白。實(shí)驗(yàn)證明Omp25蛋白抑制LPS/R848誘導(dǎo)的巨噬細(xì)胞產(chǎn)生IL-12。對其機(jī)制的研究表明Omp25上調(diào)THP-1中的mi R-155、mi R-23b和mi R-21,這些micro RNA可能參與調(diào)控Omp25對LPS/R848誘導(dǎo)的巨噬細(xì)胞IL-12產(chǎn)生的抑制作用;Omp25可以抑制LPS/R848誘導(dǎo)的THP-1中NF-κB信號途徑,證明Omp25在轉(zhuǎn)錄水平抑制THP-1表達(dá)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

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

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