【作者】 王明桥；

【导师】 夏宁邵；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 禽流感(Avian influenza, AI)是由A型流感病毒引起的烈性传染病。全球自2003年以来,已有10个国家先后报告发现人感染H5N1病例,导致170多名患者死亡,人类防控行动不可懈怠。在禽流感病毒编码的各种蛋白中,以血凝素(HA)基因的突变率最高。而且HA是流感病毒最主要的表面抗原,它能够诱导机体产生相应的中和抗体。本实验室制备的H5N1抗体库中,已发现有4株抗体对目前所检测的33株不同遗传变异亚系的H5N1病毒代表株均能有效中和,提示H5N1病毒HA上可能存在重要的高保守性中和位点。本实验利用其中的1个保守中和单抗8H5,应用噬菌体展示技术筛选HA中和表位模拟肽,为禽流感病毒血凝素蛋白(HA)高保守性中和表位的研究和疫苗的研制奠定基础。利用保守中和单抗8H5,对噬菌体随机12肽库进行筛选,得到11个噬菌体阳性克隆:122、123、124、125、126、128、129、130、131、132、133。这些12肽序列在HA蛋白上没有找到同源性很高的氨基酸序列,说明这些12肽可能是在空间结构上模拟了HA的中和表位构象。为了进一步研究12肽与抗体8H5的结合活性,以及各12肽免疫小鼠产生抗体的性质分析,对12肽进行了融合蛋白的表达。将12肽以单拷贝和双拷贝两种方式与239蛋白C端融合表达,通过间接法ELISA和竞争法ELISA对融合蛋白与抗体8H5结合活性进行检测,结果显示单拷贝融合12肽123、125和双拷贝融合12肽122、129保持与8H5单抗特异结合活性,并且这四种融合形式的多肽均对单抗8H5与病毒YU22的结合有较强的竞争抑制作用。进一步以12肽123和125为例,探索类病毒颗粒展示多肽的不同拷贝数对多肽活性以及免疫效果的影响。将多肽串联在HBcAg B细胞优势表位区段(aa.79-80),可形成35nm和30nm两种类病毒颗粒。ELISA结果显示,除以一个拷贝展示的12肽不能与8H5单抗特异结合外,12肽123和125以二、三、四、五个拷贝数展示时均保持与8H5单抗特异结合活性,但结合活性和免疫效果并没有随着多肽数的增加而得到明显加强。同时,将类病毒颗粒展示肽纯化、复性后免疫小鼠。ELISA检测结果显示免疫1周后,抗体滴度就可达到1:1000,5周后抗体滴度到达1:32000。通过免疫荧光实验,验证含有3个拷贝123肽和4个拷贝125肽的HBcAg类病毒颗粒展示肽免疫小鼠血清中,有针对流感病毒HA蛋白的特异性抗体的产生,显示了多肽成为HA模拟表位疫苗的可行性。根据12肽123和125的实验结果,将其余10个12肽均以2个拷贝的方式串联在HBcAg B细胞优势表位区段。ELISA结果显示,122、124、128和129以两个拷贝方式在类病毒颗粒上展示时,保持与8H5单抗特异结合活性。通过对以上各种形式多肽的检测及分析,确定了12肽与载体蛋白的融合表达方式以及活性影响因素——载体蛋白性质和多肽拷贝数。同时,根据不同融合蛋白的免疫效果的比较,将类病毒颗粒载体作为表位疫苗设计的理想载体模型。本研究对表位模拟肽的各种性质进行了进一步的实验分析。采用Dot blot、Western blot等方法对表位模拟肽的构象性进行分析,发现从线性12肽库中筛选出的12肽同样具有“构象依赖性”,对今后筛选其他表位提供一些参考。同时,利用实验检测和软件分析了各个12肽之间的同源性和进化方向,确定122和125,123和128,124和129分别为同一类型多肽。最后通过定点突变的研究,为HA中和表位的研究提供理论和数据的参考。总之,通过对禽流感病毒HA表位模拟肽的各种融合蛋白的表达与活性分析,免疫反应性分析,以及对表位模拟肽各种性质的实验分析,为高致病性H5N1禽流感病毒血凝素蛋白(HA)模拟表位的研究奠定了基础。更多还原

【Abstract】 Avian Influenza (AI) is one of syndrome disease of birds caused by influenza A virus in avian. At present, highly pathogenic avian influenza (HPAI) is listed in A infectious diseases by World Organization for Animal Health (OIE). The disease broke out in many countries of the world, and resulted in a serious economic loss. At the same time, it threatens the public health. The HA is the major surface antigen of influenza virus, which can induce the production of neutralizing antibodies to neutralize the infectivity of the virus. And there are five antigenic sites mapped on the HA. Antigenic changes of HA play an important role in influenza virus variation. We found four monoclonal antibodies which neutralized 34 strains of the H5N1 virus in our antibody panel. It suggested that there’s a highly conservative epitope in H5N1 virus. Thus it gives us a clue to research the epitope of HA and epitope-based vaccines, and a approach of overcoming the H5N1 avian influenza virus.In the paper, McAb 8H5 was used to screen for the mimotops from Ph.D.-12TM phage display peptide library. 11 positive phage clones were obtained: 122, 123, 124, 125, 126, 128, 129, 130, 131, 132, 133. The homologous sequence of the HA protein was not found.To assay the affinity of these peptides and McAb 8H5, and the immune response, we design a recombinant protein of corresponding peptide and 239 protein, including single-copy peptide and double-copy peptides. The results of indirect ELISA and competitive ELISA showed high affinity of McAb 8H5 and recombinant proteins, including 239 -123, 239-125, 239-D122 and 239-D124. Then, peptide 123 and 125 as an example, were used to study the affection of copy numbers to the activity of peptide displayed on HBV virus-like particle. 2, 3, 4, 5 copies peptide of 123 and 125 maintained the high affinity to McAb 8H5, except the single copy. Meanwhile, we immunized BALB/c mice with HBV virus-like particle recombinant protein. The immuno- fluorescence assay demonstrated that antibodies specific for HA were induced, and the titer of antibodies raised quickly. It showed the feasibility of mimotops as HA epitope vaccine. Further more, the other 10 peptides were designed for double copies peptide displayed on HBV virus-like particles. Another four high affinity recombinant protein to McAb 8H5 were obtained, including HBc-D122, HBc-D124, HBc-D128 and HBc-D129. There was also a high titer of antibody after immunization. Thus, we chose the HBV VLP as a peptide displaying vector for epitope vaccine.The further research on the peptides results of Dot blot and Western blot showed that the peptide from the linear peptide library should also form a specifical conformation; we called it“conformation-dependent”. Meanwhile, the analysis of homologous character of peptide mimotopes and the analysis of evolutional tree of peptide mimotopes, indicated that 122 and 125, 128 and 124, 123 and 129 were the same type of peptide respectively.In conclusion, through the assay of recombinant proteins and the analysis of peptides, we try to found the basis for the research on mimotopes of highly pathogenic H5N1 avian influenza A virus HA.更多还原