本文選題：布魯氏菌　切入點(diǎn)：IV型分泌系統(tǒng)　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：布魯氏菌(Brucella)是革蘭陰性兼性胞內(nèi)寄生菌,能夠引起布魯氏菌病(Brucellosis),簡(jiǎn)稱布病。布病是一種在家畜和人類間廣泛傳播的人畜共患傳染病,給很多國(guó)家和地方造成巨大的經(jīng)濟(jì)損失和公共安全問(wèn)題。該病屬于慢性疾病,可能持續(xù)數(shù)周或數(shù)月,如果不能被有效治療,就會(huì)引起肝臟,脾臟,淋巴結(jié),骨髓,生殖系統(tǒng)以及骨骼系統(tǒng)出現(xiàn)病理變化。胚胎滋養(yǎng)層細(xì)胞是布魯氏菌主要的靶細(xì)胞之一,已有的研究結(jié)果表明布魯氏菌在宿主細(xì)胞內(nèi)生存主要與宿主細(xì)胞的內(nèi)質(zhì)網(wǎng)(ER)緊密相關(guān)。而布魯氏IV型分泌系統(tǒng)對(duì)布魯氏菌抑制宿主天然免疫應(yīng)答和胞內(nèi)生存具有重要作用,主要是通過(guò)分泌效應(yīng)分子來(lái)實(shí)現(xiàn)的。VceC蛋白是布魯氏菌IV型分泌系統(tǒng)的效應(yīng)分子和主要毒力因子之一,揭示其在感染宿主細(xì)胞中的作用及其作用機(jī)制,對(duì)于闡明布魯氏菌胞內(nèi)生存和免疫逃逸的機(jī)制具有重要意義。本試驗(yàn)通過(guò)構(gòu)建VceC的真核表達(dá)載體并轉(zhuǎn)染山羊滋養(yǎng)層細(xì)胞(GTC),檢測(cè)(1)內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志性分子GRP78和CHOP蛋白表達(dá)量變化,未折疊蛋白反應(yīng)(UPR)信號(hào)通路的激活,以及UPR通路中IRE1蛋白表達(dá)變化,分析VceC蛋白對(duì)布魯氏菌感染過(guò)程中宿主細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)的影響;(2)山羊滋養(yǎng)層細(xì)胞上清液中分泌的孕酮和雌激素的濃度變化,探究VceC蛋白在布魯氏菌侵染GTC過(guò)程中對(duì)其性腺激素分泌的影響。試驗(yàn)結(jié)果如下:1.克隆布魯氏菌VceC基因,利用pEGFP-C1載體構(gòu)建真核表達(dá)載體pEGFP-C1-VceC,轉(zhuǎn)染GTC,利用免疫熒光和Western Blot檢測(cè)VceC蛋白,結(jié)果表明,成功構(gòu)建及轉(zhuǎn)染真核表達(dá)載體pEGFP-C1-VceC。2.以pEGFP-C1-VceC轉(zhuǎn)染GTC,通過(guò)Western Blot檢測(cè)發(fā)現(xiàn)GRP78表達(dá)在12 h和24 h均顯著升高(P0.01),而24 h后CHOP表達(dá)顯著降低(P0.05),表明Vce C蛋白能夠激發(fā)GTC的內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng);RT-PCR和Western Blot進(jìn)一步檢測(cè)UPR相關(guān)分子,結(jié)果表明,VceC蛋白激活UPR中的IRE1通路引起內(nèi)質(zhì)網(wǎng)應(yīng)激。3.ELISA檢測(cè)細(xì)胞培養(yǎng)上清液的孕酮和雌激素,VceC組孕酮含量在轉(zhuǎn)染12 h和24 h顯著低于空白組和空載體組(P0.01),雌激素?zé)o明顯差異,表明VceC蛋白影響GTC孕酮的分泌。
[Abstract]:Brucella. brucella is a gram-negative facultative intracellular parasite that can cause brucellosis. Brucellae is a zoonotic infectious disease that spreads widely between domestic animals and humans. It's a chronic disease that can last for weeks or months and can cause liver, spleen, lymph nodes, bone marrow if not treated effectively. There are pathological changes in the reproductive and skeletal systems. Embryonic trophoblastic cells are one of the main target cells of Brucella. Previous studies have shown that the survival of Brucella in host cells is closely related to the endoplasmic reticulum (ERR) of host cells, and Brucella type IV secretory system plays an important role in inhibiting innate immune response and intracellular survival of the host. VceC protein, which is mainly realized by secreting effector molecules, is one of the effector molecules and one of the main virulence factors of Brucella type IV secretory system, which reveals its role in the infection of host cells and its mechanism. It is important to elucidate the mechanism of intracellular survival and immune escape of Brucella. By constructing eukaryotic expression vector of VceC and transfecting goat trophoblastic cells with GTCU, we detected the changes of GRP78 and CHOP protein expression levels in the iconic molecules of endoplasmic reticulum stress (ER). The activation of unfolded protein response (UPR) signaling pathway and the expression of IRE1 protein in the UPR pathway were observed. Effects of VceC protein on endoplasmic reticulum stress in host cells during brucella infection the concentration of progesterone and estrogen in the supernatant of goat trophoblastic cells was analyzed. To explore the effect of VceC protein on gonadal hormone secretion during brucella infection with GTC. The results are as follows: 1. Clone the VceC gene of Brucella, construct eukaryotic expression vector pEGFP-C1-VceCby pEGFP-C1 vector, transfect it, detect VceC protein by immunofluorescence and Western Blot. The results show that. The eukaryotic expression vector pEGFP-C1-VceC.2was successfully constructed and transfected with pEGFP-C1-VceC. The results of Western Blot detection showed that the expression of GRP78 increased significantly at 12 h and 24 h, while the expression of CHOP decreased significantly after 24 h, indicating that VceC protein could stimulate the endoplasmic reticulum response of GTC. UPR related molecules were further detected by RT-PCR and Western Blot. The results showed that the activation of IRE1 pathway in UPR induced endoplasmic reticulum stress by Elisa. The levels of progesterone and progesterone in the culture supernatant of VceC group were significantly lower than those in the blank group and empty vector group at 12 h and 24 h after transfection, and there was no significant difference in estrogen between the two groups. The results showed that VceC protein affected the secretion of GTC progesterone.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.27
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本文編號(hào)：1639631