人14型和55型腺病毒中和抗体表位分析

发布时间：2018-03-26 09:41

  本文选题：14型腺病毒　切入点：55型腺病毒　出处：《广州医科大学》2017年硕士论文

【摘要】：1.研究背景人14型腺病毒(Ad14)及55型腺病毒(Ad55)均属B种腺病毒,近年来常有爆发,可致重症呼吸道疾病甚至死亡。Ad14与Ad55具有高度同源的Penton base和Fiber蛋白,但Hexon蛋白高变区(Hyper-Viable-Region,HVR)差异较大。前期结果显示,我国人群Ad14、Ad55群体免疫水平较低,普遍面临其威胁。目前尚无抗Ad14及Ad55的特效药物或预防疫苗,分析Ad14、Ad55的主要中和表位有利于治疗性抗体及预防性疫苗的研发。利用改换表面蛋白或部分片段的嵌合病毒可定位中和抗体的优势表位。2.研究目的制备携带Ad55单个HVR的嵌合Ad14报告病毒,分析人中和血清识别的主要中和表位,研究自然感染与疫苗免疫诱导的抗体的表位识别特性,为单克隆中和抗体及疫苗研发提供参考。3.研究方法基于实验室前期构建的E3基因敲除的Ad14,通过同源重组技术将其Hexon高变区HVR1至HVR7分别替换为Ad55的相应HVR,得到携带Ad55 HVR的嵌合Ad14报告病毒;检测Ad14阳性血清、Ad55阳性血清对这些嵌合病毒的中和活性,分析人中和抗体的优势表位;利用Western-blot技术及Fiber蛋白吸附技术研究Fiber特异性抗体的中和活性;利用Westerin-blot技术分析Penton特异性抗体的产生情况;以灭活Ad14、Ad55免疫小鼠制备小鼠中和血清,研究小鼠抗体的表位识别谱;以Fiber蛋白免疫小鼠,探索Fiber蛋白诱导交叉中和抗体的能力。4.研究结果成功制备7种携带Ad55单个HVR及1种携带全部Ad55 HVR的嵌合Ad14报告病毒,发现人源Hexon特异性中和抗体主要识别HVR2,5,7。Fiber特异性中和抗体仅存在于Ad14,Ad55双阳性血清,且对Ad14、Ad55均具有中和活性。双阳性血清、Ad14单阳性血清、Ad55单阳性血清均含有Penton结合抗体,但不具备中和活性。小鼠免疫血清主要识别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蛋白可诱导显著的交叉中和抗体反应,而且Ad55 Fiber蛋白具有较强的免疫源性。5.结论阐明自然感染和疫苗免疫诱导的Ad14、Ad55中和抗体的表位识别特性,发现Fiber是交叉中和抗体的主要靶标,且以其免疫小鼠可诱导交叉中和抗体反应。这些发现为研究Ad14和Ad55的免疫学特性提供新的线索,也为单克隆中和抗体研发及疫苗设计奠定基础。
[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.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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