鼠衣原体质粒缺失株的构建及体外传代对其生物学特性和致病性的影响

发布时间：2018-03-12 06:53

本文选题：鼠衣原体　切入点：质粒　出处：《南华大学》2015年博士论文　论文类型：学位论文

【摘要】：衣原体是一类严格真核细胞内寄生、具有独特发育周期的原核细胞型微生物,可引起人和动物多种疾病,疾病表现复杂。对人致病的衣原体有沙眼衣原体、肺炎衣原体和鹦鹉热衣原体等。衣原体感染已成为当今世界具有挑战性的重大公共卫生问题和社会问题,对健康和经济有着巨大的影响。为有效应对衣原体感染给公共卫生所带来的挑战,加强衣原体基础研究,深入研究其致病机制、研制衣原体疫苗成为世界范围内衣原体研究学者的重要研究课题。目前,鼠衣原体作为一种模式菌已广泛应用于人类病原性衣原体致病机制研究和衣原体疫苗研究中。一、鼠衣原体质粒缺失株的构建和鉴定背景与目的:衣原体质粒在衣原体致病中发挥重要作用,利用质粒缺失菌株和质粒携带菌株进行比较研究,可以观察研究质粒的遗传学功能,此外质粒缺失菌株还可以用作基因工程菌,广泛应用于分子生物学研究;因此构建和鉴定衣原体质粒缺失株具有重要意义。方法:采用新生霉素处理鼠衣原体、噬斑形成实验结合以Pgp3为检测标志的间接免疫荧光分析方法筛选鼠衣原体质粒缺失株,进一步采用PCR检测质粒基因,碘染色观察糖原聚集现象,动物试验检测其致病性。结果:在96孔微孔板中进行的筛选试验中,筛选到5个质粒缺失菌株。通过PCR分析证实这些菌株缺乏质粒基因,此外,在上述菌株感染的HeLa细胞中没有发现GlgA蛋白的分泌现象,也无糖原聚集。质粒缺失菌株CMUT3经阴道感染C3H/HeJ小鼠未见诱导小鼠输卵管积水。结论:成功构建了鼠衣原体质粒缺失株。二、体外传代对鼠衣原体生物学特性和致病性的影响背景与目的:尽管鼠衣原体在实验室已传代数十年,有关传代选择作用于鼠衣原体的结局尚未见报道。最近,我们对鼠衣原体质粒缺失株cmut3和野生型菌株进行传代培养,采用“无辅助感染和辅助感染”交替的方式进行体外传代培养,然后观察传代的菌株感染细胞时对离心辅助感染因素的依赖性;传代的cmg28和cmut3g40在感染hela细胞时对离心这种辅助感染因素的依赖性降低。c3h/hej小鼠经阴道感染鼠衣原体野生型菌株cmg0和传代的cmg28,阴道感染cmg28后引起的输卵管积水病理变化与cmg0感染组相比,明显降低。因此,传代的鼠衣原体在体内的致病性与体外细胞感染特性之间存在一个较大的差异。本实验的目的就是来探寻这种矛盾后面存在的可能机制。方法:体外传代培养鼠衣原体野生型菌株和质粒缺失株至一定代数,提取基因组dna,通过下一代测序技术对cmg0、cmg28、cmut3g5和cmut3g40进行全基因组测序,比较分析基因组数据;同时动物试验检测鼠衣原体的致病性。结果:通过对cmg0和cmg28进行深度基因组分析,cmg28和cmg0的基因组在3个开放读码框中存在显著差异:tc0237(q117e)替代突变(cmg28,100%;cmg0,0%),tc0668(g216*)无义突变(6.4%vs0%)和tc0668(g322r)替代突变(26%vs0%),和多个的tc0412突变多态性。吸附试验表明cmg28对细胞的吸附能力增强。cmg28和cmg0经阴道感染c3h/hej小鼠后,二者在小鼠体内有着相似的上行感染能力,而cmg28感染组致输卵管积水发生率明显降低;bio-plex200系统检测显示感染cmg28后小鼠输卵管组织中炎性细胞因子显著减少,这与输卵管炎症细胞浸润显著减少相关。tc0237(q117e)是cmg28和cmut3g40共有的突变,提示tc0237(q117e)与鼠衣原体体外吸附能力增强相关。结论:鼠衣原体TC0237(Q117E)突变增强体外吸附能力。鼠衣原体TC0237(Q117E)/TC0668(G322R)突变与鼠衣原体的致病性减弱相关联。
[Abstract]:Chlamydia is a strictly parasitic in eukaryotic cells, prokaryotic microorganism has a unique developmental cycle, can cause human and animal diseases, disease complex. Of human pathogenic Chlamydia have chlamydia, Chlamydia pneumoniae and Chlamydia psittaci. Chlamydia infection has become a major public health problem and challenge social problems, has great influence on health and economy. In order to effectively deal with Chlamydia infection to public health challenges, strengthen the basic research of Chlamydia, in-depth study of its pathogenic mechanism, vaccine research garment corpus has become an important research topic in the world Chlamydia research scholars. At present, the rat as a model bacterium Chlamydia has been widely used in human pathogenic and pathogenic mechanism of Chlamydia trachomatis vaccine research. A construction and identification of Chlamydia trachomatis induced mutant plasmid Background and purpose: will play an important role in the Chlamydia trachomatis plasmid carrying strains were pathogenic in comparative studies using plasmid deficient strain and plasmid, genetic function can be observed in addition plasmid, the plasmid deficient strain can also be used as gene engineering bacteria, widely used in molecular biology research; therefore the construction and identification of Chlamydia plasmid deficient strain has important significance methods: the novobiocin treatment in Chlamydia, plaque formation experiment combined with indirect immunofluorescence to detect Pgp3 markers analysis method for screening mutant rat Chlamydia plasmid, further detected by PCR gene plasmid, observe the phenomenon of glycogen accumulation of iodine staining, animal test their pathogenicity. Results: the screening test was carried out in 96 micro holes in 5, by screening the plasmid deficient strain. By PCR analysis indicated that these strains lacking plasmid gene, in addition, in the above The phenomenon of secretion of GlgA protein found no strain infected HeLa cells, no glycogen accumulation. The plasmid deficient strain CMUT3 vaginal infection C3H/HeJ mice no mice induced by hydrosalpinx. Conclusion: the successful construction of rat Chlamydia plasmid deletion strains. Two, in vitro on rat kinuhara body biological characteristics and pathogenic effects of background with the objective: although Chlamydia trachomatis induced in the laboratory has been passed algebra for ten years, the passage selection in the rat Chlamydia outcome has not been reported. Recently, we have to pass Daipei rat trachomatis plasmid mutant cmut3 and wild-type strain, with no auxiliary auxiliary infection and infection "alternate ways were cultured in vitro. Then to observe the infection factors of centrifugal auxiliary dependence when passaged strains infected cells; the passage cmg28 and cmut3g40 in HeLa cells infected with the infection factors of the auxiliary centrifugal Dependent decrease in.C3h/hej mice by Chlamydia trachomatis induced wild type strain cmg0 and cmg28 were vaginal infection, vaginal infection caused by cmg28 after fallopian tube pathology and cmg0 infection group, significantly reduced. Therefore, the passage of rat Chlamydia has a large difference in pathogenicity in vivo and in vitro cell. The infection characteristics the purpose of the experiment is to explore the possible mechanism of this contradiction behind. Methods: in vitro cultured rat Chlamydia wild-type strain and mutant plasmid to a certain algebra, genomic DNA was extracted by next-generation sequencing technology on cmg0, cmg28, cmut3g5 and cmut3g40 for whole genome sequencing, comparative genome analysis and pathogenic data; detection of Chlamydia trachomatis induced animal test. Results: through in-depth analysis of cmg0 genome and cmg28 genome, cmg28 and cmg0 in 3 open reading frames are significant The difference: tc0237 (q117e) mutation (cmg28100%; cmg0,0%), tc0668 (g216*) nonsense mutation (6.4%vs0%) and tc0668 (g322r) mutation (26%vs0%), mutation and multiple tc0412 polymorphism. The adsorption test showed that the adsorption capacity of cmg28 cells enhanced.Cmg28 and cmg0 by c3h/hej after vaginal infection in mice two, have the similar ability of ascending infection in mice, while cmg28 infection group significantly reduced the incidence of hydrosalpinx caused by fallopian; bio-plex200 detection showed cmg28 infection mouse oviduct inflammatory cytokines in tissues was significantly reduced, and the fallopian tube inflammatory cell infiltration was significantly reduced.Tc0237 (q117e) is a common mutation cmg28 and cmut3g40, suggesting that tc0237 (q117e) associated with the adsorption capacity of rat in vitro enhanced. Conclusion: Chlamydia trachomatis TC0237 rats (Q117E) in vitro. TC0237 mutation enhances the adsorption capacity of rat Chlamydia (Q117E) mutation and /TC0668 (G322R) The pathogenicity of Chlamydia is associated with the decrease of Chlamydia.

【学位授予单位】：南华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R374

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