發(fā)布時間：2018-03-26 09:41

  本文選題：14型腺病毒　切入點：55型腺病毒　出處：《廣州醫(yī)科大學》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。前期結果顯示,我國人群Ad14、Ad55群體免疫水平較低,普遍面臨其威脅。目前尚無抗Ad14及Ad55的特效藥物或預防疫苗,分析Ad14、Ad55的主要中和表位有利于治療性抗體及預防性疫苗的研發(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)勢表位。2.研究目的制備攜帶Ad55單個HVR的嵌合Ad14報告病毒,分析人中和血清識別的主要中和表位,研究自然感染與疫苗免疫誘導的抗體的表位識別特性,為單克隆中和抗體及疫苗研發(fā)提供參考。3.研究方法基于實驗室前期構建的E3基因敲除的Ad14,通過同源重組技術將其Hexon高變區(qū)HVR1至HVR7分別替換為Ad55的相應HVR,得到攜帶Ad55 HVR的嵌合Ad14報告病毒;檢測Ad14陽性血清、Ad55陽性血清對這些嵌合病毒的中和活性,分析人中和抗體的優(yōu)勢表位;利用Western-blot技術及Fiber蛋白吸附技術研究Fiber特異性抗體的中和活性;利用Westerin-blot技術分析Penton特異性抗體的產(chǎn)生情況;以滅活Ad14、Ad55免疫小鼠制備小鼠中和血清,研究小鼠抗體的表位識別譜;以Fiber蛋白免疫小鼠,探索Fiber蛋白誘導交叉中和抗體的能力。4.研究結果成功制備7種攜帶Ad55單個HVR及1種攜帶全部Ad55 HVR的嵌合Ad14報告病毒,發(fā)現(xiàn)人源Hexon特異性中和抗體主要識別HVR2,5,7。Fiber特異性中和抗體僅存在于Ad14,Ad55雙陽性血清,且對Ad14、Ad55均具有中和活性。雙陽性血清、Ad14單陽性血清、Ad55單陽性血清均含有Penton結合抗體,但不具備中和活性。小鼠免疫血清主要識別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蛋白可誘導顯著的交叉中和抗體反應,而且Ad55 Fiber蛋白具有較強的免疫源性。5.結論闡明自然感染和疫苗免疫誘導的Ad14、Ad55中和抗體的表位識別特性,發(fā)現(xiàn)Fiber是交叉中和抗體的主要靶標,且以其免疫小鼠可誘導交叉中和抗體反應。這些發(fā)現(xiàn)為研究Ad14和Ad55的免疫學特性提供新的線索,也為單克隆中和抗體研發(fā)及疫苗設計奠定基礎。
[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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392

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本文編號：1667357