【作者】 颜渊清；

【导师】 张军；

【作者基本信息】 厦门大学 ， 细胞生物学， 2007， 硕士

【摘要】 H5N1禽流感病毒已经在亚洲,欧洲和非洲广泛地传播,不仅造成了巨大的经济损失,而且这种病毒已经感染到人类,导致人员的大量伤亡。随着病毒在禽类的连续性爆发,通过基因重配或者基因突变,这种病毒有可能在人与人间传播。据预测,如果H5N1病毒成为下一次全球性爆发的病毒株,由于人没有或者几乎没有对这种病毒株的免疫性,将会有上亿人死于这种病毒。WHO呼吁全球各个国家都要有应对这种病毒的策略。最近本实验室鉴定出3株对多种来源的H5N1代表株均有良好中和活性的识别H5亚型血凝素（HA）的广谱单克隆抗体8H5、8G9和10F7,对于寻找克服禽流感高变性的广谱治疗性抗体、疫苗和药物具有重要价值。本研究应用分子模拟技术,将克隆得到的3株广谱单抗采用“典范结构”方法进行结构模建,模型结构在cvff力场下进行能量最小化和分子动力学模拟的优化,并通过能量、“拉曼强传图”检验、Profiles-3D分析等理论验证,获得较为合理的3株抗体Fab的三维空间结构。将3株单抗Fab分子依次与已有晶体结构的3个H5亚型HA分子（PDB编号是1jsm、2ibx和2fk0）进行分子对接,利用分子对接结果分析H5亚型禽流感的广谱中和表位和针对中和表位的药物设计的多肽参考物,其中综合单个Fab分子与3个HA分子的对接结果用于寻找该单抗识别的HA抗原表位,用于寻找与抗原良好契合的多肽结构。综合分析分子对接的结果表明:（1）HA蛋白的Fab8H5表位是由9个不连续氨基酸残基组成的:（以1jsm氨基酸编号计）Asp⁶⁸, Asn⁷²(,Glu¹¹², Lys¹¹³, Ile¹¹⁴), Pro¹¹⁸, Ser¹²⁰, Tyr¹³⁷, Tyr²⁵²;（2）HA蛋白的10F7表位也是由9个不连续的氨基酸残基所组成:Asn⁷²,(Glu¹¹²,Lys¹¹³,Ile¹¹⁴),(Ser¹²⁰,Ser¹²¹),Arg¹⁴⁵,Tyr¹⁶⁴,Tyr²⁵²;（3）HA蛋白的8G9表位位于Glu¹¹²到Ser¹²⁰之间的区域;（4）Fab8H5上的Tyr⁵⁰-Ser⁵¹-Ser⁵²-Asn⁵³-Leu⁵⁴-Ala⁵⁵-Pro⁵⁶多肽结构, Fab8G9上的Gly⁹⁵-Ieu⁹⁶-Ala⁹⁷-Thr⁹⁸-Leu⁹⁹-Met¹⁰⁰-Val^100a-Leu^100b-Pro^100c-Asp¹⁰¹-Tyr¹⁰²多肽结构,Fab10F7上的Ala²⁶-Tyr²⁷-Thr²⁸-Phe²⁹-Thr³⁰-Ser³¹-Tyr³²和Gly⁹⁵-Gly⁹⁶-Thr⁹⁷-Gly⁹⁸ -Asp⁹⁹-Phe¹⁰⁰-His^100a-Tyr^100b-Ala^100c-Met^100d-Asp⁽101(-Tyr¹⁰²多肽结构,可作为针对HA中和表位药物设计的多肽结构参考物。所有这些结果将为H5亚型禽流感病毒广谱疫苗和治疗性药物的分子设计提供依据,为禽流感感染和中和机制以及受体研究提供有用的参考信息。更多还原

【Abstract】 The H5N1 avian influenza virus has widely spread in Asia, Europe and Africa, causing a large amount of economic loss. Moreover, the virus has infected human, resulting in lots of human died. With the spreading of the virus in birds, the human-by-human infection may become possible if the gene recombination and mutation occurs constantly. It is predicted that there will be more than 100 million human dead because of little or lack of immunity against the virus if the next pandemic breaks out by H5N1. WHO calls on each country to design the strategies for the prevention of the virus. Our research group have found three strains of broad neutral monoclonal antibodies, named 8H5, 8G9 and 10F7 respectively, all of which bind to various H5N1 virus strains well. These monoclonal antibodies are helpful in seeking the broad curative antibodies, developing the vaccine and designing the anti-virus drug against the high mutational H5N1 influenza virus.In this study, the structures of the three antibodies were modeled by the“canonical structure”method, and then the models were subjected to energy minimization in cvff force filed. A lot of verification test programs, such as energy analysis, Ramachandran plot and Profiles-3D were employed to make sure the structures were in good conformation.The three antibody models were subject to docking with three HA structures found in PDB （PDB ID: 1jsm, 2ibx and 2fk0）. From the docking results, we analyzed the binding pattern between the antibodies and the HAs. The binding pattern between the individual antibody with the three HAs was used to search the broad neutral epitopes which the three antibodies binding to and the interacting polypeptides located in the antibodies which may be made use of the reference of the anti-virus drug.From the docking results analysis, we got the following conclusion: （1） The common epitopes of the three HAs against Fab8H5 were comprised as followings: （the residue in 1jsm） Asp⁶⁸, Asn⁷², (Glu¹¹², Lys¹¹³, Ile¹¹⁴), Pro¹¹⁸, Ser¹²⁰, Tyr¹³⁷, Tyr²⁵². （2） The common epitopes against Fab10F7 were: Asn⁷², (Glu¹¹², Lys¹¹³, Ile¹¹⁴), (Ser¹²⁰,Ser¹²¹), Arg¹⁴⁵, Tyr⁽164(,Tyr²⁵². （3） The epitopes against Fab8G9 located in the region Glu¹¹² to Ser¹²⁰. （4） The polypeptide located in Fab8H5 which was Tyr⁵⁰-Ser⁵¹-Ser⁵² -Asn⁽53(-Leu⁵⁴-Ala⁵⁵-Pro⁵⁶, the polypeptide located in Fab8G9 which was Gly⁹⁵-Ieu⁹⁶ -Ala⁹⁷-Thr⁹⁸-Leu⁹⁹-Met¹⁰⁰-Val^100a-Leu^100b-Pro^100c-Asp¹⁰¹-Tyr¹⁰², and the polypeptides located in Fab10F7 which were Ala²⁶-Tyr²⁷-Thr²⁸-Phe²⁹ -Thr³⁰-Ser³¹-Tyr³² and Gly⁹⁵-Gly⁹⁶-Thr⁹⁷-Gly⁹⁸-Asp⁹⁹-Phe¹⁰⁰-His^100a-Tyr⁽100b(-Ala^100c -Met^100d-Asp¹⁰¹-Tyr¹⁰² may be used as the references for the anti-virus drugs.The results will provide the valuable information to the development of the broad vaccine and to the design of the curative drug against H5 influenza virus, and the important reference for the research in the mechanic of virus infection and neutralization.更多还原