【摘要】： 志賀氏菌屬(Shigella spp.)細菌是一類不形成芽孢的革蘭氏陰性致病菌,能夠通過侵襲大腸引起患者典型菌痢癥狀(發(fā)熱、腹痛、腹瀉)。根據(jù)生化反應(yīng)和O-抗原結(jié)構(gòu)的不同,志賀氏菌可以分為四個群。其中,福氏志賀氏菌和宋內(nèi)志賀氏菌比鮑氏志賀氏菌和痢疾志賀氏菌分布更廣。據(jù)WHO統(tǒng)計,每年約有1.647億人感染痢疾,110萬人死亡,大多數(shù)為5歲以下的兒童。 福氏志賀氏菌(S. flexneri)是發(fā)展中國家主要的感染菌株,2a血清是主要的血清型。近幾年來我國每年大約有2000萬人次感染痢疾,年累計發(fā)病數(shù)一直位于第3位,僅次于肺結(jié)核和乙肝。由于志賀氏菌感染劑量極低(10-100個細菌),主要傳播途徑為糞口途徑,所以最常見的擴散形式是人與人之間的傳播,加之多重耐藥菌株的不斷出現(xiàn),常規(guī)的抗生素治療方式遇到了巨大的挑戰(zhàn)。由于人們對痢疾桿菌的致病機理和宿主的免疫保護機制還不十分清楚,所以迄今為止仍未研究出能有效控制痢疾的理想疫苗。 有研究表明,福氏2a志賀氏菌在37℃時發(fā)揮完整的細胞侵襲力,而在30℃時卻不能侵襲上皮細胞。本實驗室對福氏2a志賀氏菌2457T不同培養(yǎng)溫度下全菌蛋白的比較蛋白質(zhì)組研究發(fā)現(xiàn):當(dāng)溫度升高到37℃時,GlpQ、GlpB蛋白表達上調(diào)。而當(dāng)福氏2a志賀氏菌2457T毒力大質(zhì)粒缺失時,GlpQ、GlpB蛋白表達也顯著上調(diào)。顯然,GlpQ、GlpB蛋白對福氏2a志賀氏菌2457T的毒力有影響。而GlpQ、GlpB蛋白由GlpR、DeoR、H-NS三個蛋白調(diào)控,H-NS蛋白的研究已有報道,后邊也有討論,而GlpR、DeoR蛋白屬于DeoR蛋白家族,它們是一種細菌性調(diào)節(jié)蛋白(PF00455)。DeoR是一種轉(zhuǎn)錄調(diào)節(jié)子,阻遏脫氧核糖的代謝。GlpR也是一種轉(zhuǎn)錄調(diào)節(jié)子,調(diào)節(jié)3-磷酸甘油的阻遏。 為了探索福氏2a志賀氏菌2457T中g(shù)lpR、deoR基因的功能,本研究將由Datsenko和Wanner建立的λ噬菌體Red重組系統(tǒng)稍加改進,成功地敲除了福氏2a志賀氏菌2457T株的glpR、deoR基因,并利用低拷貝質(zhì)粒構(gòu)建了回復(fù)體加以驗證,對GlpR、DeoR進行了初步的功能研究。對野生株、突變株與回復(fù)株的生長曲線、生化反應(yīng)進行了比較分析,結(jié)果表明,野生株、突變株和回復(fù)株的生長曲線基本一致;突變株與野生株的生化反應(yīng)沒有明顯差異。通過豚鼠角膜實驗、HeLa細胞模型和小鼠動物模型檢測glpR、deoR基因缺失對志賀氏菌毒力的影響,發(fā)現(xiàn)野生株、突變株與回復(fù)株均能夠引起豚鼠角膜強烈炎癥反應(yīng),而glpR基因的缺失會明顯提高志賀氏菌的毒力。利用雙向電泳和質(zhì)譜技術(shù)對野生株、突變株和回復(fù)株進行了全菌蛋白的比較蛋白質(zhì)組學(xué)研究。在蛋白質(zhì)組的比較分析中,我們發(fā)現(xiàn),glpR、deoR基因敲除和回復(fù)后,表達上調(diào)的蛋白點共有14個,表達下調(diào)的有3個。結(jié)果表明,由于glpR、deoR基因都是轉(zhuǎn)錄抑制子,它們的缺失,能使其抑制的一系列蛋白表達上調(diào)。缺失glpR基因后,甘油激酶(GlpK)、3-磷酸甘油脫氫酶B亞單位(GlpB)和磷酸二酯酶(GlpQ)表達上調(diào)。而缺失deoR后,胸苷磷酸化酶(DeoA)、嘌呤核苷磷酸(DeoD)、2 -脫氧核糖- 5 -磷酸醛縮酶(DeoC)等表達上調(diào)。 YciD (NP-836950)是含有212個氨基酸殘基的,分子量為22kDa的小分子量假想外膜蛋白,屬于OmpW家族蛋白,該蛋白的功能未知。本實驗室在福氏2a志賀菌2457T野生株與hns(又稱virR)基因缺失突變株蛋白表達譜的比較研究中發(fā)現(xiàn),37℃培養(yǎng)條件下,hns基因缺失突變株中外膜蛋白YciD表達量下調(diào),我們認為該蛋白可能受H-NS的調(diào)控。為探索福氏2a志賀菌2457T中yciD基因的功能,本文采用改進的λ-Red重組系統(tǒng)成功構(gòu)建了該基因的缺失突變體。
[Abstract]:Shigella spp. ) The bacteria are a class of Gram-negative pathogenic bacteria that do not form a spore, and can cause typical bacillary dysentery (fever, abdominal pain, and diarrhea) caused by the invasion of the large intestine. Shigella can be divided into four groups according to the biochemical reaction and the O-antigen structure. Among them, Shigella flexneri and Shigella sonneae are more widely distributed than Shigella flexneri and Shigella in Shigella. According to the WHO statistics, about 164.7 million people are infected with diarrhea each year, and 1.1 million people die, and most of them are children under the age of 5. Shigella flexneri (S. flexneri) is the main infection strain in developing countries, and 2a serum is the main Serotype. In recent years, about 20 million people have been infected with dysentery per year, and the cumulative incidence of the year has been in the third place, second only to tuberculosis. As a result of the extremely low dose of Shigella (10-100 bacteria), the main transmission route is the faecal route, so the most common form of diffusion is the spread of human and human, and the continuous appearance of multiple drug-resistant strains, and the conventional antibiotic treatment method has encountered a great deal. The challenge is that the mechanism of the disease and the mechanism of the immune protection of the host are not very clear so far, the reason for the effective control of the dysentery has not been studied so far The results of the study showed that the 2-a-shigella flexneri exhibited a complete cell-invasion force at 37 & deg; C, while at 30 鈩，

本文編號：2460730