本文選題：布魯氏菌 + 載脂蛋白（ApoBR）��； 參考：《石河子大學(xué)》2014年碩士論文

【摘要】：布魯氏菌病是由布魯氏菌(Brucella)引起的一種人畜共患傳染病，人感染布病后的臨床癥狀主要表現(xiàn)為波浪熱；動物感染后主要為流產(chǎn)、睪丸炎等癥狀。布魯氏菌可長期寄生在巨噬細(xì)胞內(nèi)并抑制感染細(xì)胞的凋亡，引起人、畜的多器官損傷和慢性感染，給人類健康和畜牧業(yè)發(fā)展帶來嚴(yán)重威脅。因此，對布魯氏病的發(fā)病機(jī)制進(jìn)行研究，尋找有效的治療性藥物和有效的疫苗迫在眉睫。 目的：在本實驗室黃小強(qiáng)篩選到與布魯氏菌OMP25互作的宿主蛋白為ApoBR，其在布魯氏菌侵染巨噬細(xì)胞中的作用并不清楚，為了了解ApoBR的作用而展開研究。本文通過RNAi等技術(shù)探討ApoBR在布魯氏菌侵染巨噬細(xì)胞中的作用，為布魯氏菌逃避巨噬細(xì)胞殺傷作用的機(jī)制和抑制腫瘤壞死因子信號通路方面的調(diào)節(jié)作用奠定基礎(chǔ)。 方法：（1）應(yīng)用生物信息學(xué)技術(shù)將ApoBR蛋白的基因應(yīng)用siDESIGN Center網(wǎng)頁設(shè)計干擾片段并合成，將合成的干擾片段連接到干擾載體pSIREN-RetroQ-ZsGreen上，構(gòu)建10個干擾載體，通過qRT-PCR對干擾載體進(jìn)行初篩，篩選出抑制效果較為明顯的RNAi干擾片段。（2）將初篩獲得的RNAi干擾片段，構(gòu)建RNAi慢病毒載體pLentiLox3.7-X（X分別為干擾片段ApoBR-253、ApoBR-893、ApoBR-1325和ApoBR-2053），進(jìn)一步篩選出更高效的RNAi干擾片段。（3）用牛種布魯氏菌S2308侵染高效轉(zhuǎn)染RNAi慢病毒載體的小鼠巨噬細(xì)胞，在4h、8h、12h、24h收集細(xì)胞和上清，，細(xì)胞用裂解液裂解，梯度稀釋，進(jìn)行胞內(nèi)CFU計數(shù)，上清用于ELISA檢測腫瘤壞死因子(TNF-α)的變化。裂解侵染的轉(zhuǎn)染慢病毒載體的巨噬細(xì)胞，提取RNA，應(yīng)用qRT-PCR檢測促凋亡因子Bax和抑制凋亡因子Bcl-2的變化，并用流式細(xì)胞儀檢測布魯氏菌侵染的轉(zhuǎn)染慢病毒細(xì)胞的凋亡率。 結(jié)果：(1)測序結(jié)果表明成功構(gòu)建了10個干擾載體pSIREN-RetroQ-ZsGreen-X（X分別為ApoBR-163、ApoBR-457、ApoBR-648、ApoBR-2668、ApoBR-1138、 ApoBR-893、ApoBR-253、ApoBR-1325、ApoBR-2053、CK）。應(yīng)用qRT-PCR技術(shù)和生物信息學(xué)分析比對成功篩選出4個比較高效的干擾載體pSIREN-RetroQ-ZsGreen-X（X分別為：ApoBR-253、ApoBR-893、ApoBR-1325、ApoBR-2053）。（2）成功構(gòu)建pLentiLox3.7-253、 pLentiLox3.7-893、pLentiLox3.7-1325、pLentiLox3.7-20534個慢病毒載體，并篩選獲得2個更高效的干擾載體pLentiLox3.7-253和pLentiLox3.7-1325，抑制率高達(dá)60%以上（3）布魯氏菌2308侵染高效轉(zhuǎn)染慢病毒載體的小鼠巨噬細(xì)胞RAW264.7的CFU結(jié)果與對照組和陰性對照組相比CFU降低。ELISA檢測TNF-α結(jié)果顯示與對照組和陰性對照組相比均呈下降趨勢。流式細(xì)胞儀檢測細(xì)胞的凋亡率結(jié)果顯示，與對照組和陰性對照組相比，凋亡率均升高，RT-PCR檢測Bax、Bcl-2的結(jié)果顯示，促凋亡因子Bax相對表達(dá)量與對照組和陰性對照組相比均升高，而抑制凋亡因子Bcl-2的結(jié)果卻相反。 結(jié)論：本實驗結(jié)果表明ApoBR是布魯氏菌侵染巨噬細(xì)胞的重要識別受體之一，對布魯氏菌的入侵和早期感染具有重要的作用；使用靶向ApoBR的RNAi慢病毒載體能有效抑制布魯氏菌在巨噬細(xì)胞中的增殖，ApoBR的表達(dá)導(dǎo)致TNF-a和Bax上調(diào)，Bcl-2下調(diào)，細(xì)胞凋亡提高，這為激活天然免疫反應(yīng)在抗布魯氏菌提供了一個靶位點，為尋找對布魯氏菌病有效的治療性藥物提供了科學(xué)依據(jù)。
[Abstract]:Brucellosis is a zoonosis caused by Brucella (Brucella). The main clinical symptoms of brucellosis are wave heat. After infection, the main symptoms are abortion and orchitis. Brucella can parasitism in macrophages and inhibit the apoptosis of infected cells for a long time, causing multiple organ damage in human and animal. And chronic infection poses a serious threat to the development of human health and animal husbandry. Therefore, it is imminent to study the pathogenesis of brucellosis and to find effective therapeutic drugs and effective vaccines.
Objective: in our laboratory Huang Xiaoqiang screened the host protein of Brucella OMP25 interacting with Brucella ApoBR, and its role in the infection of Brucella in macrophages was not clear. In order to understand the role of ApoBR, the role of ApoBR in the infection of Brucella to macrophages was explored by RNAi and other techniques to escape from Brucella. The mechanism of macrophage killing and the regulation of tumor necrosis factor signaling pathway lay the foundation.
Methods: (1) using bioinformatics technology to use the gene of ApoBR protein to design interference fragments and synthesize the siDESIGN Center web page, connect the interfered fragments to the interference carrier pSIREN-RetroQ-ZsGreen, construct 10 interference carriers, screen the interference carrier through qRT-PCR, and screen out the RNAi interference with more obvious inhibition effect. (2) the RNAi lentivirus vector pLentiLox3.7-X (X, ApoBR-253, ApoBR-893, ApoBR-1325 and ApoBR-2053) was constructed by screening the RNAi interference fragment of the initial screening, and the more efficient RNAi interference fragments were further screened. (3) the mouse macrophages infected with the Brucella bovine S2308 were infected by the high efficiency transfection of RNAi lentivirus vector. 24h collected cells and supernatants, the cells were lysed with lysate, gradient dilution, intracellular CFU count, and the supernatant was used to detect the changes of tumor necrosis factor (TNF- alpha) by ELISA. The macrophages transfected with lentivirus vector transfected by lysate were RNA, and qRT-PCR was used to detect the changes of apoptotic factor Bax and the inhibition of apoptosis factor Bcl-2, and flow cytometry was used. The apoptosis rate of lentivirus transfected by Brucella was detected.
Results: (1) the results of sequencing showed that 10 interference carriers pSIREN-RetroQ-ZsGreen-X (X, ApoBR-163, ApoBR-457, ApoBR-648, ApoBR-2668, ApoBR-1138, ApoBR-893, ApoBR-253, ApoBR-1325, ApoBR-2053, CK) were successfully constructed, and 4 more efficient interference carriers were successfully screened by application and bioinformatics analysis. TroQ-ZsGreen-X (X: ApoBR-253, ApoBR-893, ApoBR-1325, ApoBR-2053). (2) successful construction of pLentiLox3.7-253, pLentiLox3.7-893, pLentiLox3.7-1325, pLentiLox3.7-20534 lentivirus vector, and screening 2 more efficient interference carrier pLentiLox3.7-253 and pLentiLox3.7-1325, inhibition rate up to 60% (3) Brucella 2308 The CFU results of mouse macrophage RAW264.7 infected with high efficiency transfection of lentivirus vector compared with the control group and the negative control group, the result of CFU decreased.ELISA detection TNF- alpha showed a decreasing trend compared with the control group and the negative control group. The apoptosis rate of the flow cytometry showed that the apoptosis was compared with the control group and the negative control group. The rate of Bax was detected by RT-PCR, and the results of Bcl-2 showed that the relative expression of Bax was higher than that of the control group and the negative control group, but the result of the inhibition of the apoptotic factor Bcl-2 was the opposite.
Conclusion: the results show that ApoBR is one of the important recognition receptors for Brucella infection in macrophages, which plays an important role in the invasion and early infection of Brucella; the use of the RNAi lentivirus vector targeting ApoBR can effectively inhibit the proliferation of Brucella in macrophages. The expression of ApoBR leads to the up regulation of TNF-a and Bax, and Bcl-2 under Bcl-2. The enhancement of cell apoptosis, which provides a target site for the activation of natural immune response to Brucella, provides a scientific basis for the search for effective therapeutic drugs for brucellosis.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R516.7

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