本文選題：登革病毒 + 寨卡病毒��； 參考：《廣州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景2014年廣東省爆發(fā)了1型登革熱疫情,2015年出現(xiàn)了2型登革病毒(DENV2)流行。2016年,隨著寨卡病毒(ZIKV)在美洲地區(qū)爆發(fā)流行,中國也發(fā)現(xiàn)了輸入性病例。目前認(rèn)為ADE效應(yīng)是重癥登革發(fā)生的重要風(fēng)險因素,但DENV或ZIKV感染后宿主的免疫狀況仍不清。登革病毒共有四種血清型(DENV1-4),同一血清型因病毒株序列的差異又可被分為不同的基因型。我們的前期研究發(fā)現(xiàn)2014年爆發(fā)的登革熱疫情中有兩種不同的基因型同時流行,不同的基因型間是否存在血清抗體的交叉反應(yīng)?新發(fā)的ZIKV感染常與DENV出現(xiàn)在同一地區(qū)。ZIKV在序列和結(jié)構(gòu)上與DENV有高度同源性,其中DEVN2 E蛋白序列與ZIKV一致性高達54%,這種高度同源性可引起免疫學(xué)上的交叉反應(yīng)。研究內(nèi)容及目的本文首先探討DENV1血清型不同基因型感染機體后血清抗體的產(chǎn)生,揭示登革熱同一血清型不同基因型間血清抗體交叉反應(yīng)的變化;并進一步以2015年潮州地區(qū)DENV2病人及本院收治的ZIKA病人為研究對象,分析登革熱和寨卡病毒誘導(dǎo)宿主抗體產(chǎn)生及交叉反應(yīng)的特征。研究方法1.DENV1,2型及ZIKV重組E蛋白質(zhì)粒的構(gòu)建及蛋白表達C端帶標(biāo)簽的DENV1(GenotypeⅣ和GenotypeⅠ)、DENV2和ZIKA重組E蛋白的表達采用哺乳動物細(xì)胞表達體系。從病人急性期血清提取病毒RNA,用RT-PCR法或兩步PCR法擴增病毒E蛋白膜外區(qū),克隆到質(zhì)粒pc DNA 3.1構(gòu)建表達質(zhì)粒。經(jīng)序列驗證的質(zhì)粒轉(zhuǎn)染293T細(xì)胞,收獲含重組E蛋白的細(xì)胞上清,直接進行下一步捕獲ELISA。2.捕獲ELISA的建立用抗標(biāo)簽的抗體捕獲293T細(xì)胞轉(zhuǎn)染上清中的E蛋白,建立捕獲ELISA法。3.血清抗體與重組病毒E蛋白結(jié)合分析1)用建立的ELISA法檢測36例DENV1感染者病程不同時期血清與GenotypeⅣ、GenotypeⅠE蛋白的結(jié)合反應(yīng),血清樣品以1:1000稀釋開始,10倍系列稀釋,共5個稀釋度。2)30例DENV2感染者和3例ZIKV感染者血清抗體分別與ZIKV,DENV1和DENV2E蛋白結(jié)合反應(yīng)。血清樣品以1:100稀釋開始,3倍系列稀釋,共8個稀釋度。研究結(jié)果1.成功構(gòu)建ZIKV,DENV1和DENV2重組E蛋白表達質(zhì)粒,并經(jīng)序列驗證,表達蛋白大小約56KD。以表達的E蛋白建立了可用于血清抗體檢測的捕獲ELISA法。2.在檢測的36例DENV1型病例中,病程不同時期的血清與GenotypeⅣ和GenotypeⅠE蛋白均有明顯的結(jié)合反應(yīng),在病程后期血清表現(xiàn)更強的抗體結(jié)合反應(yīng)(P=0.038)3.在檢測的30例DENV2型住院病例中,大部分病例(24/30)血清與DENV1和ZIKV E蛋白有明顯的結(jié)合反應(yīng),表現(xiàn)為3種不同的結(jié)合形式,DENV1ZIKA(4/30),DENV1ZIKA(11/30)和DENV=ZIKA(9/30)。在檢測的3例ZIKV血清中,有兩例在急性期及恢復(fù)期均呈很強的抗體反應(yīng),且與DENV1和DENV2有明顯交叉。結(jié)論1.成功在哺乳動物細(xì)胞體系表達重組E蛋白,并建立了可用于血清抗體檢測的捕獲ELISA法。2.DENV1血清型不同基因型間均有抗體交叉結(jié)合反應(yīng),且隨病程結(jié)合反應(yīng)有增加的趨勢。3.DENV和ZIKV均可誘導(dǎo)宿主血清抗體的產(chǎn)生,且呈高度的交叉反應(yīng)特征。
[Abstract]:Background there was a dengue type 1 outbreak in Guangdong province in 2014 and a dengue virus type 2 (DENV2) epidemic in 2015. In 2016, with the outbreak of Zika virus ZIKV in the Americas, imported cases were also found in China. ADE effect is considered to be an important risk factor for severe dengue, but the host immune status after DENV or ZIKV infection is still unclear. There are four serotypes of dengue virus (DENV1-4), and the same serotype can be divided into different genotypes because of the different sequences of the virus strains. Our previous study found that there were two different genotypes in the dengue outbreak in 2014. Are there cross-reactions between different genotypes? The new ZIKV infection often appears in the same region as DENV. ZIKV has high homology with DENV in sequence and structure, and the sequence of DEVN2 E protein is as high as 54% with ZIKV. This high homology can cause cross-reaction in immunology. Objective to investigate the production of serum antibodies after infection with different genotypes of DENV1 serotypes, and to reveal the changes of serum antibody cross-reactions among different genotypes of dengue fever. The characteristics of host antibody production and cross reaction induced by dengue fever and Zika virus were analyzed with DENV2 patients and ZIKA patients in Chaozhou area in 2015. Methods 1. Construction of recombinant E protein particles and expression of C-terminal tagged DENV1(Genotype 鈪，

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