ApoBR在布鲁氏菌胞内寄生中的功能研究

发布时间：2018-05-12 04:38

本文选题：布鲁氏菌 + 载脂蛋白（ApoBR）　；参考：《石河子大学》2014年硕士论文

【摘要】：布鲁氏菌病是由布鲁氏菌(Brucella)引起的一种人畜共患传染病，人感染布病后的临床症状主要表现为波浪热；动物感染后主要为流产、睾丸炎等症状。布鲁氏菌可长期寄生在巨噬细胞内并抑制感染细胞的凋亡，引起人、畜的多器官损伤和慢性感染，给人类健康和畜牧业发展带来严重威胁。因此，对布鲁氏病的发病机制进行研究，寻找有效的治疗性药物和有效的疫苗迫在眉睫。目的：在本实验室黄小强筛选到与布鲁氏菌OMP25互作的宿主蛋白为ApoBR，其在布鲁氏菌侵染巨噬细胞中的作用并不清楚，为了了解ApoBR的作用而展开研究。本文通过RNAi等技术探讨ApoBR在布鲁氏菌侵染巨噬细胞中的作用，为布鲁氏菌逃避巨噬细胞杀伤作用的机制和抑制肿瘤坏死因子信号通路方面的调节作用奠定基础。方法：（1）应用生物信息学技术将ApoBR蛋白的基因应用siDESIGN Center网页设计干扰片段并合成，将合成的干扰片段连接到干扰载体pSIREN-RetroQ-ZsGreen上，构建10个干扰载体，通过qRT-PCR对干扰载体进行初筛，筛选出抑制效果较为明显的RNAi干扰片段。（2）将初筛获得的RNAi干扰片段，构建RNAi慢病毒载体pLentiLox3.7-X（X分别为干扰片段ApoBR-253、ApoBR-893、ApoBR-1325和ApoBR-2053），进一步筛选出更高效的RNAi干扰片段。（3）用牛种布鲁氏菌S2308侵染高效转染RNAi慢病毒载体的小鼠巨噬细胞，在4h、8h、12h、24h收集细胞和上清，，细胞用裂解液裂解，梯度稀释，进行胞内CFU计数，上清用于ELISA检测肿瘤坏死因子(TNF-α)的变化。裂解侵染的转染慢病毒载体的巨噬细胞，提取RNA，应用qRT-PCR检测促凋亡因子Bax和抑制凋亡因子Bcl-2的变化，并用流式细胞仪检测布鲁氏菌侵染的转染慢病毒细胞的凋亡率。结果：(1)测序结果表明成功构建了10个干扰载体pSIREN-RetroQ-ZsGreen-X（X分别为ApoBR-163、ApoBR-457、ApoBR-648、ApoBR-2668、ApoBR-1138、 ApoBR-893、ApoBR-253、ApoBR-1325、ApoBR-2053、CK）。应用qRT-PCR技术和生物信息学分析比对成功筛选出4个比较高效的干扰载体pSIREN-RetroQ-ZsGreen-X（X分别为：ApoBR-253、ApoBR-893、ApoBR-1325、ApoBR-2053）。（2）成功构建pLentiLox3.7-253、 pLentiLox3.7-893、pLentiLox3.7-1325、pLentiLox3.7-20534个慢病毒载体，并筛选获得2个更高效的干扰载体pLentiLox3.7-253和pLentiLox3.7-1325，抑制率高达60%以上（3）布鲁氏菌2308侵染高效转染慢病毒载体的小鼠巨噬细胞RAW264.7的CFU结果与对照组和阴性对照组相比CFU降低。ELISA检测TNF-α结果显示与对照组和阴性对照组相比均呈下降趋势。流式细胞仪检测细胞的凋亡率结果显示，与对照组和阴性对照组相比，凋亡率均升高，RT-PCR检测Bax、Bcl-2的结果显示，促凋亡因子Bax相对表达量与对照组和阴性对照组相比均升高，而抑制凋亡因子Bcl-2的结果却相反。结论：本实验结果表明ApoBR是布鲁氏菌侵染巨噬细胞的重要识别受体之一，对布鲁氏菌的入侵和早期感染具有重要的作用；使用靶向ApoBR的RNAi慢病毒载体能有效抑制布鲁氏菌在巨噬细胞中的增殖，ApoBR的表达导致TNF-a和Bax上调，Bcl-2下调，细胞凋亡提高，这为激活天然免疫反应在抗布鲁氏菌提供了一个靶位点，为寻找对布鲁氏菌病有效的治疗性药物提供了科学依据。
[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.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.7

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