本文選題：胸膜肺炎放線(xiàn)桿菌　切入點(diǎn)：CpxAR　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：胸膜肺炎放線(xiàn)桿菌(Actinobacillus pleuropneumoniae,APP)是一種具有高度傳染性的呼吸道病原體,以纖維素性壞死性支氣管肺炎和纖維素性胸膜炎為特征,可引起豬傳染性胸膜肺炎,給全球養(yǎng)豬業(yè)帶來(lái)巨大的經(jīng)濟(jì)損失。有研究表明,在多個(gè)菌種中Cpx AR雙組份調(diào)控系統(tǒng)在調(diào)控細(xì)菌毒力作用方面發(fā)揮著重要功能,并且參與生物被膜形成以及耐藥性機(jī)制,但是其在APP中的功能尚不清楚。本文通過(guò)研究Cpx AR基因缺失株,研究了Cpx AR在APP中對(duì)于生物被膜形成、耐藥性以及毒力功能上的作用。1.雙組分調(diào)控系統(tǒng)Cpx AR基因缺失株的鑒定與生長(zhǎng)曲線(xiàn)測(cè)定Cpx AR基因缺失株及其互補(bǔ)菌株由本實(shí)驗(yàn)室保存,通過(guò)PCR及基因測(cè)序驗(yàn)證了其正確性,在不同培養(yǎng)基中的生長(zhǎng)曲線(xiàn)通過(guò)每小時(shí)OD600的值得以測(cè)定,通過(guò)嚴(yán)謹(jǐn)反應(yīng)試驗(yàn)結(jié)果證實(shí)了Cpx AR對(duì)APP的應(yīng)激耐受能力發(fā)揮作用。2.基因缺失株與野生株生物被膜形成能力和耐藥性的比較在生物被膜形成能力的檢測(cè)試驗(yàn)中,通過(guò)96孔板結(jié)晶紫染色法比較了野生株S4074,突變株ΔCpx AR以及互補(bǔ)菌株CΔCpx AR三者形成生物被膜能力的差異,結(jié)果證實(shí)了缺失Cpx AR后,APP形成生物被膜的能力明顯降低。同時(shí),通過(guò)比較三種菌株對(duì)不同抗生素的MIC值,比較了其耐藥性上的差異。3.Cpx AR調(diào)控APP生物被膜形成的分子機(jī)制研究通過(guò)熒光定量PCR試驗(yàn)發(fā)現(xiàn)了受Cpx AR調(diào)控的生物被膜相關(guān)基因。利用電泳遷移分析法和DNaseⅠ足跡法發(fā)現(xiàn),Cpx AR可以直接調(diào)控rpo E基因的轉(zhuǎn)錄,而不是hns,來(lái)調(diào)節(jié)pga操縱子的功能,進(jìn)而影響生物被膜的表達(dá)。4.基因缺失株與野生株毒力差異的比較通過(guò)小鼠存活曲線(xiàn)試驗(yàn)和組織載菌量試驗(yàn),證實(shí)了缺失Cpx AR后,APP的毒力發(fā)生了下降,并且突變株在肺部組織中的攜帶能力明顯低于野生株和互補(bǔ)菌株。
[Abstract]:Actinobacillus pleuropneumoniae (APP) is a highly infectious respiratory pathogen characterized by cellulose necrotizing bronchopneumonia and cellulose pleurisy. Some studies have shown that the Cpx AR two-component regulatory system plays an important role in regulating the virulence of bacteria and is involved in the formation of biofilm and the mechanism of drug resistance. But its function in APP is not clear. In this paper, we studied the effect of Cpx AR on biofilm formation in APP by studying Cpx AR gene deletion strain. Identification and growth curve of Cpx AR gene deletion strain and its complementary strains were preserved by our laboratory. The correctness of Cpx AR gene deletion strain was verified by PCR and gene sequencing. Growth curves in different media were measured by OD600 per hour. The stress tolerance ability of Cpx AR to APP was confirmed by rigorous reaction test. 2.Compared with that of wild strain and missing strain, the ability of biofilm formation and drug resistance were detected in the test of biofilm formation ability. The biofilm formation ability of wild strain S4074, mutant 螖 Cpx AR and complementary strain C 螖 Cpx AR was compared by 96 hole plate crystal violet staining. The results showed that the ability of app to form biofilm was significantly decreased after the absence of Cpx AR. By comparing the MIC values of three strains to different antibiotics, The molecular mechanism of CpxAR regulating the formation of APP biofilm was compared. By fluorescence quantitative PCR assay, genes related to biofilm regulated by Cpx AR were found. Electrophoresis migration analysis and DNase 鈪，

本文編號(hào)：1650366