布鲁氏菌VceC蛋白对山羊滋养层细胞内质网应激反应和性腺激素分泌的影响

发布时间：2018-03-20 15:12

  本文选题：布鲁氏菌　切入点：IV型分泌系统　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：布鲁氏菌(Brucella)是革兰阴性兼性胞内寄生菌,能够引起布鲁氏菌病(Brucellosis),简称布病。布病是一种在家畜和人类间广泛传播的人畜共患传染病,给很多国家和地方造成巨大的经济损失和公共安全问题。该病属于慢性疾病,可能持续数周或数月,如果不能被有效治疗,就会引起肝脏,脾脏,淋巴结,骨髓,生殖系统以及骨骼系统出现病理变化。胚胎滋养层细胞是布鲁氏菌主要的靶细胞之一,已有的研究结果表明布鲁氏菌在宿主细胞内生存主要与宿主细胞的内质网(ER)紧密相关。而布鲁氏IV型分泌系统对布鲁氏菌抑制宿主天然免疫应答和胞内生存具有重要作用,主要是通过分泌效应分子来实现的。VceC蛋白是布鲁氏菌IV型分泌系统的效应分子和主要毒力因子之一,揭示其在感染宿主细胞中的作用及其作用机制,对于阐明布鲁氏菌胞内生存和免疫逃逸的机制具有重要意义。本试验通过构建VceC的真核表达载体并转染山羊滋养层细胞(GTC),检测(1)内质网应激标志性分子GRP78和CHOP蛋白表达量变化,未折叠蛋白反应(UPR)信号通路的激活,以及UPR通路中IRE1蛋白表达变化,分析VceC蛋白对布鲁氏菌感染过程中宿主细胞内质网应激反应的影响;(2)山羊滋养层细胞上清液中分泌的孕酮和雌激素的浓度变化,探究VceC蛋白在布鲁氏菌侵染GTC过程中对其性腺激素分泌的影响。试验结果如下:1.克隆布鲁氏菌VceC基因,利用pEGFP-C1载体构建真核表达载体pEGFP-C1-VceC,转染GTC,利用免疫荧光和Western Blot检测VceC蛋白,结果表明,成功构建及转染真核表达载体pEGFP-C1-VceC。2.以pEGFP-C1-VceC转染GTC,通过Western Blot检测发现GRP78表达在12 h和24 h均显著升高(P0.01),而24 h后CHOP表达显著降低(P0.05),表明Vce C蛋白能够激发GTC的内质网应激反应;RT-PCR和Western Blot进一步检测UPR相关分子,结果表明,VceC蛋白激活UPR中的IRE1通路引起内质网应激。3.ELISA检测细胞培养上清液的孕酮和雌激素,VceC组孕酮含量在转染12 h和24 h显著低于空白组和空载体组(P0.01),雌激素无明显差异,表明VceC蛋白影响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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.27
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本文编号：1639631