本文選題：14型腺病毒　切入點(diǎn)：55型腺病毒　出處：《廣州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：1.研究背景人14型腺病毒(Ad14)及55型腺病毒(Ad55)均屬B種腺病毒,近年來常有爆發(fā),可致重癥呼吸道疾病甚至死亡。Ad14與Ad55具有高度同源的Penton base和Fiber蛋白,但Hexon蛋白高變區(qū)(Hyper-Viable-Region,HVR)差異較大。前期結(jié)果顯示,我國(guó)人群Ad14、Ad55群體免疫水平較低,普遍面臨其威脅。目前尚無(wú)抗Ad14及Ad55的特效藥物或預(yù)防疫苗,分析Ad14、Ad55的主要中和表位有利于治療性抗體及預(yù)防性疫苗的研發(fā)。利用改換表面蛋白或部分片段的嵌合病毒可定位中和抗體的優(yōu)勢(shì)表位。2.研究目的制備攜帶Ad55單個(gè)HVR的嵌合Ad14報(bào)告病毒,分析人中和血清識(shí)別的主要中和表位,研究自然感染與疫苗免疫誘導(dǎo)的抗體的表位識(shí)別特性,為單克隆中和抗體及疫苗研發(fā)提供參考。3.研究方法基于實(shí)驗(yàn)室前期構(gòu)建的E3基因敲除的Ad14,通過同源重組技術(shù)將其Hexon高變區(qū)HVR1至HVR7分別替換為Ad55的相應(yīng)HVR,得到攜帶Ad55 HVR的嵌合Ad14報(bào)告病毒;檢測(cè)Ad14陽(yáng)性血清、Ad55陽(yáng)性血清對(duì)這些嵌合病毒的中和活性,分析人中和抗體的優(yōu)勢(shì)表位;利用Western-blot技術(shù)及Fiber蛋白吸附技術(shù)研究Fiber特異性抗體的中和活性;利用Westerin-blot技術(shù)分析Penton特異性抗體的產(chǎn)生情況;以滅活A(yù)d14、Ad55免疫小鼠制備小鼠中和血清,研究小鼠抗體的表位識(shí)別譜;以Fiber蛋白免疫小鼠,探索Fiber蛋白誘導(dǎo)交叉中和抗體的能力。4.研究結(jié)果成功制備7種攜帶Ad55單個(gè)HVR及1種攜帶全部Ad55 HVR的嵌合Ad14報(bào)告病毒,發(fā)現(xiàn)人源Hexon特異性中和抗體主要識(shí)別HVR2,5,7。Fiber特異性中和抗體僅存在于Ad14,Ad55雙陽(yáng)性血清,且對(duì)Ad14、Ad55均具有中和活性。雙陽(yáng)性血清、Ad14單陽(yáng)性血清、Ad55單陽(yáng)性血清均含有Penton結(jié)合抗體,但不具備中和活性。小鼠免疫血清主要識(shí)別Ad14的HVR1、2、7,Ad55的HVR1、2,以及Fiber。在小鼠中,多次免疫Ad14或Ad55的Fiber蛋白可誘導(dǎo)顯著的交叉中和抗體反應(yīng),而且Ad55 Fiber蛋白具有較強(qiáng)的免疫源性。5.結(jié)論闡明自然感染和疫苗免疫誘導(dǎo)的Ad14、Ad55中和抗體的表位識(shí)別特性,發(fā)現(xiàn)Fiber是交叉中和抗體的主要靶標(biāo),且以其免疫小鼠可誘導(dǎo)交叉中和抗體反應(yīng)。這些發(fā)現(xiàn)為研究Ad14和Ad55的免疫學(xué)特性提供新的線索,也為單克隆中和抗體研發(fā)及疫苗設(shè)計(jì)奠定基礎(chǔ)。
[Abstract]:1. Human adenovirus type 14 (Ad14) and adenovirus 55 (Ad55) belong to species B adenovirus. In recent years, human adenovirus type 14 (Ad14) and adenovirus type 55 (Ad55) have frequently erupted, causing severe respiratory diseases and even death. Ad14 has high homology with Penton base and Fiber proteins with Ad55. However, the difference of Hyper-Viable-Regiona-HVRs in the hypervariable region of Hexon protein was significant. The previous results showed that the immune level of Ad14A1-Ad55 population in China was low, and it was generally threatened. There were no special drugs or preventive vaccines against Ad14 and Ad55. The analysis of the main neutralizing epitopes of Ad14 + Ad55 is beneficial to the development of therapeutic antibodies and prophylactic vaccines. The neutralizing epitope of neutralizing antibodies can be located by using chimeric viruses with modified surface proteins or partial fragments. Objective to prepare a single Ad55 carrier. Chimeric Ad14 of HVR reports viruses, To analyze the main neutralizing epitopes recognized by human neutralizing serum and to study the epitope recognition characteristics of antibodies induced by natural infection and vaccine immunization. To provide reference for the development of monoclonal neutralizing antibody and vaccine. Methods based on the pre-laboratory construction of E3 gene knockout Ad14, the HVR1 from its Hexon hypervariable region to HVR7 was replaced with the corresponding Ad55 by homologous recombination technique. The chimeric Ad14 of Ad55 HVR reported the virus. The neutralization activity of Ad14 positive serum and Ad55 positive serum to these chimeric viruses was detected, and the dominant epitopes of human neutralizing antibody were analyzed. The neutralization activity of Fiber specific antibody was studied by Western-blot technique and Fiber protein adsorption technique. Westerin-blot technique was used to analyze the production of Penton specific antibodies, mice neutralized serum was prepared by inactivating Ad14 + Ad55 to study the epitope recognition spectrum of mouse antibody, mice were immunized with Fiber protein. To explore the ability of Fiber protein to induce cross-neutralizing antibodies. The results showed that seven chimeric Ad14 reporter viruses carrying Ad55 single HVR and one chimeric Ad14 carrying all Ad55 HVR were successfully prepared. It was found that the human Hexon specific neutralizing antibody mainly recognized HVR2 + 57.Fiber specific neutralizing antibody only existed in the double positive serum of Ad14 + Ad55, and had neutralizing activity on Ad14 + Ad55. All the single positive sera of double positive serums of Ad14 and Ad14 contained Penton binding antibodies. But it has no neutralization activity. The mouse immune serum mainly recognizes HVR1O2A55-HVR1P2and Fiber. in mice, the Fiber protein of Ad14 or Ad55 can induce a significant cross-neutralizing antibody reaction. And Ad55 Fiber protein has strong immunogenic. 5.Conclusion to elucidate the epitope recognition characteristics of Ad14Ad55 neutralizing antibody induced by natural infection and vaccine immunization, it is found that Fiber is the main target of cross neutralizing antibody. These findings provide a new clue for the study of immunological characteristics of Ad14 and Ad55, and also lay a foundation for the development of monoclonal neutralizing antibodies and vaccine design.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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