【摘要】：維氏氣單胞菌(Aeromonas veronii,AV)是一種人-獸-魚共患病原菌,由于近年來分離率逐漸升高,已逐漸引起人們的廣泛關(guān)注與重視。有研究報道稱細菌在環(huán)境中大多以生物被膜形式存在,該形式是一種同浮游態(tài)相對應的生長方式,在一定程度上增強了細菌對外界環(huán)境的抵抗力,而且細菌生物被膜態(tài)可釋放游離菌從而導致感染的遷延不愈和反復發(fā)作。若是能夠有效預防和控制生物被膜的感染,將會充分降低細菌致病的幾率,這在臨床感染治療中有著十分重要的意義。因此,篩選維氏氣單胞菌浮游態(tài)與生物被膜態(tài)間的差異蛋白,在探討維氏氣單胞菌生物被膜形成機理以及抑制生物被膜形成的用藥靶點的篩選等方面具有一定意義。本研究以維氏氣單胞菌TH0426為研究對象,首先應用96孔微量板定量檢測法探究在不同的條件下體外生物被膜形成能力,并對其條件進行優(yōu)化。結(jié)果顯示,維氏氣單胞菌TH0426在LB、THB以及TSB培養(yǎng)基中培養(yǎng),生物被膜形成能力差異不顯著;培養(yǎng)基pH為6、Nacl濃度6g/L放置于20℃培養(yǎng)箱中培養(yǎng)24h時,生物被膜形成能力較佳。同時運用掃描電子顯微鏡進一步檢測了生物被膜形成情況。在此基礎(chǔ)上,應用iTRAQ技術(shù)分析浮游態(tài)與生物被膜態(tài)下維氏氣單胞菌TH0426蛋白表達的差異,并應用MRM技術(shù)進行驗證。結(jié)果顯示,浮游態(tài)細菌與生物被膜態(tài)下細菌相比,蛋白表達存在一定的差異,共獲得277個差異蛋白,其中上調(diào)蛋白130個,下調(diào)蛋白147個,同時對這些蛋白進行了初步的生物信息學分析。本研究又挑選5個差異蛋白進行MRM驗證;同時隨機挑選上調(diào)和下調(diào)蛋白共20個,應用熒光定量PCR進行驗證,結(jié)果與iTRAQ結(jié)果相同。研究最后選出上調(diào)蛋白基因glpD,利用同源重組技術(shù)構(gòu)建維氏氣單胞菌glpD基因缺失株,并通過比較野生株、缺失株的生物被膜形成能力:發(fā)現(xiàn)glpD基因缺失株的生物被膜的形成能力較野生株有所下降。應用K-B紙片法檢測缺失株與野生株耐藥情況,變化不顯著。研究結(jié)果有助于從蛋白水平上深化維氏氣單胞菌生物被膜免疫機理及致病機理的研究,并為其在疫苗生產(chǎn)及藥物研發(fā)等發(fā)面提供科學依據(jù)。
[Abstract]:Aeromonas Vickers (Aeromonas veronii,AV) is a zoonotic pathogen, which has attracted more and more attention due to the increasing isolation rate in recent years. It has been reported that bacteria mostly exist in the form of biofilm in the environment, which is a growth mode corresponding to the planktonic state, which to some extent enhances the resistance of bacteria to the outside environment. Moreover, bacterial biofilm can release free bacteria and lead to prolonged infection and repeated attacks. If the biofilm infection can be effectively prevented and controlled, the probability of bacterial pathogenicity will be reduced, which is of great significance in the treatment of clinical infection. Therefore, the screening of differential proteins between planktonic and biofilm states of Aeromonas Vickers has a certain significance in exploring the mechanism of biofilm formation of Aeromonas Vickers and the screening of drug targets for inhibiting biofilm formation. In this study, the ability of biofilm formation in vitro under different conditions was investigated by 96 hole microplate quantitative detection method with Aeromonas Vickers TH0426 as the research object, and the conditions of the biofilm formation were optimized. The results showed that the ability of biofilm formation of Aeromonas Vickers TH0426 cultured in LB,THB and TSB medium was not significant. The ability of biofilm formation was better when the culture medium pH was 6g/L with a concentration of 6 渭 mol / L, and the culture medium was cultured in a incubator at 20 鈩，

本文編號：2376128