绵羊布鲁氏菌侵染小鼠巨噬细胞过程的荧光表征与分析

发布时间：2018-04-21 16:57

本文选题：布鲁氏菌16M + 布鲁氏菌M5　；参考：《石河子大学》2014年硕士论文

【摘要】：目的：构建稳定表达GFP的整合型重组布鲁氏菌16M和M5（以下简称GFP-布鲁氏菌16M和M5）；比较GFP-布鲁氏菌16M和M5与正常布鲁氏菌16M和M5在巨噬细胞中的存活能力，证明GFP基因的转入不会影响后续实验的进行。分析GFP-布鲁氏菌16M和M5进入细胞后与宿主细胞中的溶酶体、内质网、高尔基体结合产生的荧光强度；分析GFP-布鲁氏菌16M和M5侵染小鼠巨噬细胞的数量；对侵染初期与胞内溶酶体、内质网、高尔基体结合的时间测定，为布鲁氏菌在细胞内的生存繁殖及其分子机制研究提供理论参考。方法:将pMC-221-GFP载体电转化进入布鲁氏菌16M和M5感受态细胞，稳定传代后获得GFP-布鲁氏菌16M和M5。将GFP-布鲁氏菌16M和M5与正常布鲁氏菌16M和M5培养至对数期收集菌体，，用麦氏比浊法至所需浓度，按照细菌和细胞比例为100：1进行侵染，利用平板计数法检测其在胞内的存活能力；激光共聚焦显微镜观察胞内GFP-布鲁氏菌与溶酶体，内质网和高尔基体之间的结合，并检测出GFP-布鲁氏菌16M和M5与溶酶体、内质网和高尔基体结合后产生的荧光强度；用流式细胞仪测定侵染初期GFP-布鲁氏菌进入细胞及与胞内溶酶体、内质网、高尔基体初次结合的时间。结果：(1)成功获得整合重组型GFP-布鲁氏菌16M和M5。（2）GFP-布鲁氏菌16M和M5与正常布鲁氏菌16M和M5比较发现在小鼠巨噬细胞中的存活能力并无差别。（2）GFP-布鲁氏菌16M与溶酶体、内质网（ER）和高尔基体结合初期产生的荧光强度与布鲁氏菌M5相比较并无明显差异。（3）GFP-布鲁氏菌16M和M5侵染初期小鼠巨噬细胞产生的GFP+巨噬细胞没有明显区别，但是后期差距逐渐明显。（4）GFP-布鲁氏菌16M和M5侵染初期10min进入巨噬细胞，1.5h布鲁氏菌到达溶酶体，2h布鲁氏菌同时到达内质网（ER）和高尔基体。结论：（1）GFP基因的整合重组并不会影响布鲁氏菌16M和M5的生物特性。（2）GFP-布鲁氏菌16M和M5在侵染初期结合宿主细胞及胞内溶酶体、内质网和高尔基体的时间相同，这可能与二者同属布鲁氏菌Ⅰ型标准菌株有关。（3）GFP-布鲁氏菌到达内质网和高尔基体的时间相同，这可能与内质网和高尔基体之间的膜转移蛋白有关。（4）虽然布鲁氏菌16M和M5在侵染初期荧光强度和GFP+巨噬细胞数量上没有显著差别，但是胞内存活实验结果却恰恰相反。由此认为布鲁氏菌16M和M5进入小鼠巨噬细胞和达到溶酶体、内质网和高尔基体的能力没有区别，两者的差别还是在于胞内的生存能力，同样认为该能力也是其致病性的关键因素。
[Abstract]:Objective: to construct recombinant brucellosis 16M and M5 expressing GFP stably and compare the viability of GFP-brucella 16M and M5 with normal brucella 16M and M5 in macrophages. It is proved that the transfer of GFP gene does not affect the subsequent experiment. The fluorescence intensity of lysosome, endoplasmic reticulum and Golgi binding of GFP-brucella 16M and M5 into the host cells was analyzed, and the number of mouse macrophages infected by GFP-Brucella 16M and M5 was analyzed. The determination of the binding time of lysosomes, endoplasmic reticulum and Golgi body in the early stage of infection provides a theoretical reference for the survival and reproduction of Brucella in cells and the study of its molecular mechanism. Methods: pMC-221-GFP vector was transformed into 16M and M5 competent cells of Brucella spp. After stable passage, 16M and M5 of GFP-Brucella were obtained. GFP-brucella 16M and M5 were cultured with normal brucella 16M and M5 to collect bacteria in logarithmic phase. The bacteria were infected with Mak's turbidimetry to the required concentration and infected with 100: 1 ratio of bacteria to cells. The viability of GFP-brucella in the cell was detected by plate counting method. The binding of GFP-Brucella to lysosome, endoplasmic reticulum and Golgi body was observed by confocal laser microscope, and the fluorescence intensity of GFP-Brucella 16M and M5 combined with lysosome, endoplasmic reticulum and Golgi body was detected. Flow cytometry was used to determine the initial binding time of GFP-Brucella to cells and to lysosomes, endoplasmic reticulum and Golgi body in the early stage of infection. Results compared with normal brucellosis (16M and M5), the survival ability of recombinant GFP-brucella (16M and M5) in murine macrophages was not different from that in murine macrophages (16M and lysosomes). There was no significant difference between the fluorescence intensity produced by ER and Golgi body binding at the initial stage and that by brucella M5. There was no significant difference in GFP macrophages produced by murine macrophages from 16M and M5 infected with Brucella spp. However, in the late stage, the gap between GFP-brucella 16M and M5 was obvious. In the early stage of infection, 10min entered the macrophage and reached the lysosomal brucella for 1.5h. Brucella also reached the endoplasmic reticulum (ER) and Golgi body at the same time. Conclusion the integration and recombination of the GFP gene of 1: 1 strain does not affect the biological characteristics of 16M and M5 of Brucella brucella. The binding time of 16M and M5 to host cells and intracellular lysosomes is the same as that of endoplasmic reticulum and Golgi body in the early stage of infection. This may be related to the same time between GFP-Brucella and Golgi body in the endoplasmic reticulum (ER) and Golgi body. This may be related to the membrane transfer protein between endoplasmic reticulum and Golgi body. Although there is no significant difference in fluorescence intensity and the number of GFP macrophages between Brucella 16M and M5 at the initial stage of infection, the results of intracellular survival test are quite the opposite. It is concluded that the ability of Brucella 16M and M5 to enter mouse macrophages and reach lysosomes, endoplasmic reticulum (ER) and Golgi body are not different, but the difference between them lies in the viability of the cells. It is also believed that this ability is also a key factor in its pathogenicity.
【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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