衣原体噬菌体PhiCPG1衣壳蛋白Vp1对沙眼衣原体作用机制研究

发布时间：2017-12-28 06:01

本文关键词：衣原体噬菌体PhiCPG1衣壳蛋白Vp1对沙眼衣原体作用机制研究　出处：《天津医科大学》2016年博士论文　论文类型：学位论文

【摘要】：研究目的探究Vp1蛋白作用于沙眼衣原体并抑制其生长的机制。研究内容研究Vp1蛋白作用于沙眼衣原体后的光镜下及免疫荧光镜下的变化;研究Vp1蛋白作用于沙眼衣原体后不同时间点MEK/ERK通路p-ERK1/2蛋白的改变,ERK1和ERK2基因的改变,IL-1和IL-8炎性因子的改变;研究Vp1蛋白与阿奇霉素作用于沙眼衣原体后的区别与联系;研究Vp1蛋白作用于沙眼衣原体后三型分泌系统的改变;研究Vp1蛋白作用于沙眼衣原体后对其生长周期的影响。研究方法对构建的重组质粒菌Vp1-Pet30a(+)进行诱导表达,对表达出的Vp1蛋白进行SDS-PAGE电泳及Western Blot鉴定并纯化,纯化后的Vp1蛋白透析复性后进行蛋白定量、滤过除菌后使用。将处理后的Vp1和阿奇霉素与沙眼衣原体在Mc Coy细胞中作用,在光镜下及免疫荧光镜下观察两者对沙眼衣原体的抑制作用;提取各组细胞不同时间点的总蛋白,采用Western Blot方法检测各组之间ERK信号通路p-ERK1/2及总ERK1/2的变化情况;提取各组细胞的总RNA,采用Realtime-PCR方法检测各组之间ERK1及ERK2基因的变化情况;提取各组细胞不同时间点的培养液上清,用ELISA方法检测各组IL-1及IL-8的表达水平。应用纯化无菌的Vp1在Mc Coy细胞中作用于沙眼衣原体,同时设置未干预组和空白作为对照。培养12h、24h、36h、48h后,分别提取各组细胞的总RNA,应用Realtime-PCR方法检测沙眼衣原体内Tarp(CT456)、CT694、Inc A(CT119)、Tep P(CT875)、CT621、Cdsf(CT666)、Hct A(CT743)、Hct B(CT046)、Omc A(CT444)、Omc B(CT443)基因在不同时间点干预前后表达的不同。研究结果1、重组表达的Vp1及阿奇霉素对Mc Coy细胞内正常生长的沙眼衣原体均有抑制作用,光镜下及荧光显微镜下观察到包涵体减少,并且其减少的程度与Vp1的及阿奇霉素的浓度呈正相关。Vp1作用于沙眼衣原体后可与其结合并跟随其进行复制。2、Vp1及阿奇霉素作用于沙眼衣原体后均可引起Mc Coy细胞内MEK/ERK通路的改变,p-ERK1/2表达减少,ERK1及ERK2基因表达降低,尤以ERK1下降为显著。3、从Vp1与阿奇霉素起作用的时间来看,Vp1对沙眼衣原体的抑制作用主要发生于衣原体生长周期的后期,而阿奇霉素对沙眼衣原体的抑制作用主要发生于衣原体生长周期的早期。4、Vp1及阿奇霉素作用于沙眼衣原体后,Mc Coy细胞产生的炎症因子IL-1及IL-8减少,细胞炎症减轻。作用后,IL-1在感染后期减少明显;IL-8在Vp1干预后早期即有减少,阿奇霉素干预后感染后期减少明显。5、Vp1作用于沙眼衣原体后可导致沙眼衣原体内三型分泌系统分泌减弱。感染早期(EB侵入宿主细胞),Cdsf(CT666)表达下调,使EB对宿主细胞的感染能力下降。感染后期(即RB转为EB时期),Tarp(CT456)、CT694表达水平降低,衣原体的侵袭能力下降。Inc A(CT119)表达水平上升,衣原体同型融合增多,RB不断融合形成异常增大的网状体。Cdsf(CT666)、CT621表达下调,可使RB转化为EB减少。Tep P(CT875)在整个生长周期中表达水平均降低,提示Vp1可能干预衣原体与宿主细胞之间的信号通路传递,干扰包涵体形成。其可能还参与免疫相关基因的调控,使宿主细胞对衣原体的免疫反应增强,从而达到抑制衣原体生长的目的。6、Vp1作用于沙眼衣原体后,可导致沙眼衣原体生长周期晚期表达的基因水平下调。Hct A(CT743)、Hct B(CT046)、Omc A(CT444)、Omc B(CT443)在Vp1作用于沙眼衣原体后的晚期表达均较为干预组有所下降,提示Vp1可能通过某种机制抑制了沙眼衣原体生长周期晚期表达的基因,从而影响RB向EB的转化,抑制沙眼衣原体的增殖。结论Vp1蛋白可能通过干扰沙眼衣原体生长的后期RB转化为EB过程来抑制其生长。
[Abstract]:Objective to explore the mechanism of Vp1 protein acting on Chlamydia trachomatis and inhibiting the growth of Chlamydia trachomatis. Study on the effect of the content changes of Vp1 protein in Chlamydia trachomatis after light microscopy and immunofluorescence microscopy; change the role of Vp1 protein in Chlamydia trachomatis at different time points after MEK/ERK pathway of p-ERK1/2 protein, ERK1 and ERK2 genes, IL-1 and IL-8 of inflammatory factors change; differences and relations of Vp1 and protein azithromycin effect on Chlamydia trachomatis; study of Vp1 protein in Chlamydia trachomatis type three secretion system change; effect of Vp1 protein in Chlamydia trachomatis after the growth cycle of the. The recombinant plasmid Vp1-Pet30a (+) was induced to express. The expressed Vp1 protein was identified by SDS-PAGE electrophoresis and Western Blot. After purification, the purified Vp1 protein was regenerated and protein was quantified and filtrate after sterilization. After treatment of Vp1 and azithromycin and Chlamydia trachomatis in Mc Coy cells. The inhibitory effect of both Chlamydia trachomatis by light microscopy and immunofluorescence microscopy; extraction of total protein of cells in each group at different time points, the changes by using Western Blot method to detect the ERK signal pathway between p-ERK1/2 and ERK1/2; extraction of total RNA cells were detected by Realtime-PCR, the changes between the groups of ERK1 and ERK2 gene; extract supernatant of cells in each group at different time points, the expression level of ELISA was detected by IL-1 and IL-8. The purified aseptic Vp1 was used in the Mc Coy cells to act on Chlamydia trachomatis, and the non dry pregroup and the blank were set as the control. 24h, 36h, 12h cells, 48h, total RNA were extracted from the cells of each group, application of Realtime-PCR method in the detection of Chlamydia trachomatis (CT456), Tarp CT694, Inc A (CT119), Tep P (CT875), CT621, Cdsf (CT666), Hct A (CT743), Hct B (CT046), Omc A (CT444), Omc B (CT443) gene at different time points before and after the intervention of different expression. Results 1. Recombinant expression of Vp1 and azithromycin inhibited the growth of Chlamydia trachomatis in Mc Coy cells. The inclusion bodies were reduced under light microscope and fluorescence microscope, and the degree of reduction was positively correlated with the concentration of Vp1 and azithromycin. Vp1 can be combined with Chlamydia trachomatis and can be replicated with it. 2, Vp1 and azithromycin acting on Chlamydia trachomatis can cause changes of MEK/ERK pathway in Mc Coy cells, p-ERK1/2 expression is reduced, ERK1 and ERK2 gene expression decreases, especially ERK1 decreases. 3, from the time of Vp1 and azithromycin, the inhibitory effect of Vp1 on Chlamydia trachomatis mainly occurred at the late stage of Chlamydia growth cycle, while the inhibitory effect of azithromycin on Chlamydia trachomatis mainly occurred in the early stage of Chlamydia growth cycle. 4, Vp1 and azithromycin acted on Chlamydia trachomatis, the inflammation factor IL-1 and IL-8 produced by Mc Coy cells were reduced, and the inflammation of the cells was reduced. After the effect, IL-1 decreased markedly in the late stage of infection, and IL-8 decreased in the early stage of Vp1 after the intervention, and the infection later decreased significantly after the intervention of azithromycin. 5, the effect of Vp1 on Chlamydia trachomatis can lead to the decrease in the secretion of type three secretory system in Chlamydia trachomatis. At the early stage of infection (EB invasion of host cells), the expression of Cdsf (CT666) was downregulated, which reduced the infection ability of EB to the host cells. The expression level of Tarp (CT456) and CT694 decreased and the invasion ability of Chlamydia decreased in the late stage of infection (that is, when RB turned to EB). The expression level of Inc A (CT119) increased, the Homo fusion of Chlamydia increased and the RB fused to form an abnormal enlargement of the reticular body. The downregulation of Cdsf (CT666) and CT621 expression can reduce RB to EB. The expression level of Tep P (CT875) decreased during the whole growth cycle, suggesting that Vp1 may interfere with the signal transduction between Chlamydia and host cells, and interfere with the formation of inclusion bodies. It may also participate in the regulation of immune related genes, which can enhance the immune response of the host cells to Chlamydia, and thus inhibit the growth of Chlamydia. 6, Vp1 effect on Chlamydia trachomatis, can lead to the gene level of late expression of Chlamydia trachomatis. Hct A (CT743), Hct B (CT046), Omc A (CT444), Omc B (CT443) in the effect of Vp1 on the expression of Chlamydia trachomatis in late intervention group are decreased, suggesting that Vp1 may be a mechanism to inhibit the expression of Chlamydia trachomatis growth cycle late gene, thus affecting the conversion of EB to RB inhibition of the proliferation of Chlamydia trachomatis. Conclusion Vp1 protein may inhibit the growth of Chlamydia trachomatis by transforming RB into EB process.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R374.1

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