基于3C蛋白酶的肠道病毒71型生物发光检测方法的建立与初步应用

发布时间：2018-03-21 15:52

本文选题：肠病毒71型　切入点：生物发光成像　出处：《哈尔滨医科大学》2013年博士论文　论文类型：学位论文

【摘要】：目的:肠病毒71型(enterovirus 71,EV71)属于小RNA病毒,是人类手足口病主要病原体之一。EV71主要感染儿童,并会造成严重的神经并发症甚至死亡。EV71感染的致病机制目前尚不十分清楚,目前也没有被临床认可的疫苗和有效的治疗手段。在这里,我们介绍了一种新型的应用生物发光成像技术检测EV71感染的方法。方法:我们构建了一种能够表达融合蛋白AmNLuc(Q/G)BCLuc的报告质粒,它由分开的荧光素酶以及EV71 3C~(pro)切割位点构成。当融合蛋白被3C~(pro)切割后会恢复荧光素酶活性。结果:报告质粒AmNLuc(Q/G)BCLuc可以被3C~(pro)特异性切割并回复荧光素酶活性,其荧光强度与EV71感染量成正相关,可通过荧光素酶生物发光成像的方式实时且定量的监测细胞和小鼠中的EV71感染。应用这种方法,我们评估了Raf-1抑制剂GW5074和MEK1/2抑制剂U0126对EV71感染的抑制效果。结果发现我们所应用最高浓度的GW5074可以对体外(20μM)和体内(0.2 mg/kg)EV71感染的抑制率超过50%。Western blotting结果显示GW5074的抗EV71活性可能与其阻断MAPK通路有关。GW5074可能成为有效治疗EV71感染的候选药物。应用报告质粒AmNLuc(Q/G)BCLuc所得到的生物发光成像结果与Western blotting和组织病理学检测结果相一致。结论:这种实时的生物发光检测方法可以定量的检测抗EV71制剂的效果,对于抗EV71制剂的筛选和EV71致病机制的研究有很大帮助。
[Abstract]:Objective: Enterovirus 71 (enterovirus 71, EV71) belongs to a small RNA virus, is one of the major pathogens of HFMD mainly human.EV71 infected children, and may cause serious neurologic complications or even death of the pathogenic mechanism of.EV71 infection is not clear, there is no clinically approved vaccine and effective treatment. Here, we introduce a method for detection of EV71 infection by biological new luminescence imaging technique. Methods: We constructed a AmNLuc fusion protein expression plasmid BCLuc (Q/G) to report, it consists of separate luciferase and EV71 3C~ (pro) cleavage site. When fusion protein was 3C~ (pro) after cutting will restore the luciferase activity. Results: the reporter plasmid AmNLuc (Q/G) BCLuc by 3C~ (pro) specific cleavage and reply luciferase activity, the fluorescence intensity and EV71 infection was positively correlated with. Monitoring cells and mouse luciferase bioluminescence imaging and real-time quantitative EV71 in infection. Using this method, we evaluated the Raf-1 inhibitor GW5074 and MEK1/2 inhibitor U0126 on the inhibition effect of EV71 infection. We found that the highest concentration of GW5074 can be used for in vitro and in vivo (20 M (0.2 mg/kg)) inhibit EV71 infection rate of more than 50%.Western blotting showed GW5074 anti EV71 activity may be related to blocking MAPK pathway.GW5074 may be an effective drug candidate for the treatment of EV71 infection. The application of the reporter plasmid AmNLuc (Q/G) BCLuc obtained by bioluminescence imaging results and Western blotting and histopathological examination results. Conclusion: the effect of detection this real-time bioluminescence detection method can be used for anti EV71 agents, for the study of anti EV71 agents and screening of the pathogenic mechanism of EV71 has a great help Help.

【学位授予单位】：哈尔滨医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.5;R440

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