本文選題：布魯氏菌16M + 布魯氏菌M5��； 參考：《石河子大學(xué)》2014年碩士論文

【摘要】：目的：構(gòu)建穩(wěn)定表達(dá)GFP的整合型重組布魯氏菌16M和M5（以下簡稱GFP-布魯氏菌16M和M5）；比較GFP-布魯氏菌16M和M5與正常布魯氏菌16M和M5在巨噬細(xì)胞中的存活能力，證明GFP基因的轉(zhuǎn)入不會(huì)影響后續(xù)實(shí)驗(yàn)的進(jìn)行。分析GFP-布魯氏菌16M和M5進(jìn)入細(xì)胞后與宿主細(xì)胞中的溶酶體、內(nèi)質(zhì)網(wǎng)、高爾基體結(jié)合產(chǎn)生的熒光強(qiáng)度；分析GFP-布魯氏菌16M和M5侵染小鼠巨噬細(xì)胞的數(shù)量；對(duì)侵染初期與胞內(nèi)溶酶體、內(nèi)質(zhì)網(wǎng)、高爾基體結(jié)合的時(shí)間測定，為布魯氏菌在細(xì)胞內(nèi)的生存繁殖及其分子機(jī)制研究提供理論參考。 方法:將pMC-221-GFP載體電轉(zhuǎn)化進(jìn)入布魯氏菌16M和M5感受態(tài)細(xì)胞，穩(wěn)定傳代后獲得GFP-布魯氏菌16M和M5。將GFP-布魯氏菌16M和M5與正常布魯氏菌16M和M5培養(yǎng)至對(duì)數(shù)期收集菌體，，用麥?zhǔn)媳葷岱ㄖ了铦舛�，按照�?xì)菌和細(xì)胞比例為100：1進(jìn)行侵染，利用平板計(jì)數(shù)法檢測其在胞內(nèi)的存活能力；激光共聚焦顯微鏡觀察胞內(nèi)GFP-布魯氏菌與溶酶體，內(nèi)質(zhì)網(wǎng)和高爾基體之間的結(jié)合，并檢測出GFP-布魯氏菌16M和M5與溶酶體、內(nèi)質(zhì)網(wǎng)和高爾基體結(jié)合后產(chǎn)生的熒光強(qiáng)度；用流式細(xì)胞儀測定侵染初期GFP-布魯氏菌進(jìn)入細(xì)胞及與胞內(nèi)溶酶體、內(nèi)質(zhì)網(wǎng)、高爾基體初次結(jié)合的時(shí)間。 結(jié)果：(1)成功獲得整合重組型GFP-布魯氏菌16M和M5。（2）GFP-布魯氏菌16M和M5與正常布魯氏菌16M和M5比較發(fā)現(xiàn)在小鼠巨噬細(xì)胞中的存活能力并無差別。（2）GFP-布魯氏菌16M與溶酶體、內(nèi)質(zhì)網(wǎng)（ER）和高爾基體結(jié)合初期產(chǎn)生的熒光強(qiáng)度與布魯氏菌M5相比較并無明顯差異。（3）GFP-布魯氏菌16M和M5侵染初期小鼠巨噬細(xì)胞產(chǎn)生的GFP+巨噬細(xì)胞沒有明顯區(qū)別，但是后期差距逐漸明顯。（4）GFP-布魯氏菌16M和M5侵染初期10min進(jìn)入巨噬細(xì)胞，1.5h布魯氏菌到達(dá)溶酶體，2h布魯氏菌同時(shí)到達(dá)內(nèi)質(zhì)網(wǎng)（ER）和高爾基體。 結(jié)論：（1）GFP基因的整合重組并不會(huì)影響布魯氏菌16M和M5的生物特性。（2）GFP-布魯氏菌16M和M5在侵染初期結(jié)合宿主細(xì)胞及胞內(nèi)溶酶體、內(nèi)質(zhì)網(wǎng)和高爾基體的時(shí)間相同，這可能與二者同屬布魯氏菌Ⅰ型標(biāo)準(zhǔn)菌株有關(guān)。（3）GFP-布魯氏菌到達(dá)內(nèi)質(zhì)網(wǎng)和高爾基體的時(shí)間相同，這可能與內(nèi)質(zhì)網(wǎng)和高爾基體之間的膜轉(zhuǎn)移蛋白有關(guān)。（4）雖然布魯氏菌16M和M5在侵染初期熒光強(qiáng)度和GFP+巨噬細(xì)胞數(shù)量上沒有顯著差別，但是胞內(nèi)存活實(shí)驗(yàn)結(jié)果卻恰恰相反。由此認(rèn)為布魯氏菌16M和M5進(jìn)入小鼠巨噬細(xì)胞和達(dá)到溶酶體、內(nèi)質(zhì)網(wǎng)和高爾基體的能力沒有區(qū)別，兩者的差別還是在于胞內(nèi)的生存能力，同樣認(rèn)為該能力也是其致病性的關(guān)鍵因素。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R516.7

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