本文選題：腸病毒71型　切入點(diǎn)：生物發(fā)光成像　出處：《哈爾濱醫(yī)科大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：目的:腸病毒71型(enterovirus 71,EV71)屬于小RNA病毒,是人類手足口病主要病原體之一。EV71主要感染兒童,并會(huì)造成嚴(yán)重的神經(jīng)并發(fā)癥甚至死亡。EV71感染的致病機(jī)制目前尚不十分清楚,目前也沒有被臨床認(rèn)可的疫苗和有效的治療手段。在這里,我們介紹了一種新型的應(yīng)用生物發(fā)光成像技術(shù)檢測EV71感染的方法。方法:我們構(gòu)建了一種能夠表達(dá)融合蛋白AmNLuc(Q/G)BCLuc的報(bào)告質(zhì)粒,它由分開的熒光素酶以及EV71 3C~(pro)切割位點(diǎn)構(gòu)成。當(dāng)融合蛋白被3C~(pro)切割后會(huì)恢復(fù)熒光素酶活性。結(jié)果:報(bào)告質(zhì)粒AmNLuc(Q/G)BCLuc可以被3C~(pro)特異性切割并回復(fù)熒光素酶活性,其熒光強(qiáng)度與EV71感染量成正相關(guān),可通過熒光素酶生物發(fā)光成像的方式實(shí)時(shí)且定量的監(jiān)測細(xì)胞和小鼠中的EV71感染。應(yīng)用這種方法,我們?cè)u(píng)估了Raf-1抑制劑GW5074和MEK1/2抑制劑U0126對(duì)EV71感染的抑制效果。結(jié)果發(fā)現(xiàn)我們所應(yīng)用最高濃度的GW5074可以對(duì)體外(20μM)和體內(nèi)(0.2 mg/kg)EV71感染的抑制率超過50%。Western blotting結(jié)果顯示GW5074的抗EV71活性可能與其阻斷MAPK通路有關(guān)。GW5074可能成為有效治療EV71感染的候選藥物。應(yīng)用報(bào)告質(zhì)粒AmNLuc(Q/G)BCLuc所得到的生物發(fā)光成像結(jié)果與Western blotting和組織病理學(xué)檢測結(jié)果相一致。結(jié)論:這種實(shí)時(shí)的生物發(fā)光檢測方法可以定量的檢測抗EV71制劑的效果,對(duì)于抗EV71制劑的篩選和EV71致病機(jī)制的研究有很大幫助。
[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.

【學(xué)位授予單位】：哈爾濱醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.5;R440

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相關(guān)期刊論文 前1條

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本文編號(hào)：1644535