CpxAR双组份调控系统在胸膜肺炎放线杆菌中对生物被膜和毒力功能的影响研究

发布时间：2018-03-22 21:02

  本文选题：胸膜肺炎放线杆菌　切入点：CpxAR　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)是一种具有高度传染性的呼吸道病原体,以纤维素性坏死性支气管肺炎和纤维素性胸膜炎为特征,可引起猪传染性胸膜肺炎,给全球养猪业带来巨大的经济损失。有研究表明,在多个菌种中Cpx AR双组份调控系统在调控细菌毒力作用方面发挥着重要功能,并且参与生物被膜形成以及耐药性机制,但是其在APP中的功能尚不清楚。本文通过研究Cpx AR基因缺失株,研究了Cpx AR在APP中对于生物被膜形成、耐药性以及毒力功能上的作用。1.双组分调控系统Cpx AR基因缺失株的鉴定与生长曲线测定Cpx AR基因缺失株及其互补菌株由本实验室保存,通过PCR及基因测序验证了其正确性,在不同培养基中的生长曲线通过每小时OD600的值得以测定,通过严谨反应试验结果证实了Cpx AR对APP的应激耐受能力发挥作用。2.基因缺失株与野生株生物被膜形成能力和耐药性的比较在生物被膜形成能力的检测试验中,通过96孔板结晶紫染色法比较了野生株S4074,突变株ΔCpx AR以及互补菌株CΔCpx AR三者形成生物被膜能力的差异,结果证实了缺失Cpx AR后,APP形成生物被膜的能力明显降低。同时,通过比较三种菌株对不同抗生素的MIC值,比较了其耐药性上的差异。3.Cpx AR调控APP生物被膜形成的分子机制研究通过荧光定量PCR试验发现了受Cpx AR调控的生物被膜相关基因。利用电泳迁移分析法和DNaseⅠ足迹法发现,Cpx AR可以直接调控rpo E基因的转录,而不是hns,来调节pga操纵子的功能,进而影响生物被膜的表达。4.基因缺失株与野生株毒力差异的比较通过小鼠存活曲线试验和组织载菌量试验,证实了缺失Cpx AR后,APP的毒力发生了下降,并且突变株在肺部组织中的携带能力明显低于野生株和互补菌株。
[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，

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