高致病性禽流感病毒H5N1治疗性嵌合抗体研究

发布时间：2018-03-21 13:30

本文选题：高致病性禽流感病毒　切入点：治疗性抗体　出处：《厦门大学》2009年硕士论文　论文类型：学位论文

【摘要】： 高致病性禽流感(Avian influenza,AI)是由A型流感病毒H5N1引起的烈性传染病。近年来随着疾病的不断蔓延,禽流感病毒不仅使各地养禽业蒙受巨大损失,而且还不断突破种间屏障引发人感染事件。目前高致病性禽流感面临的问题是病死率居高不下且缺乏有效的治疗药物。由于流感病毒的持续快速变异,自然界中H5N1病毒已逐渐形成多种抗原性不同的变异亚系病毒株共流行的状况,要满足长期战略储备药物的需求,研制的H5N1治疗药物必须具备广谱性。中和单抗是防治病毒性疾病的一种有效手段,但由于禽流感病毒基因突变频率高,特别是血凝素(HA)基因,因此针对禽流感病毒的中和单抗中和谱较窄,往往只能中和少数遗传变异亚系的病毒,极大限制了这些中和单抗的应用。基于本实验室近期构建的一个具有血凝抑制(AI)活性的H5特异性单克隆抗体库(已经用41株包含全部10个遗传进化分支的H5N1病毒代表株对这些单抗的HI活性和以小鼠为动物模型的中和活性进行了测定),本研究选取四株广谱中和活性强的鼠单抗:4D1、10F7、8H5、13D4进行人源化改造,以降低鼠抗在人治疗过程中的HAMA反应,为这些抗体尽快进入临床应用奠定基础;同时也希望建立一套治疗性抗体人源化研究的平台体系,包括基因的改造、表达载体的构建、哺乳动物细胞高效稳定表达、无血清大规模培养、抗体纯化工艺等。本研究先以RT-PCR的方式从鼠杂交瘤细胞内获得四株抗体的可变区基因,再结合至人抗体IgG1的恒定区,插入pcDNA3.1表达载体瞬时表达得到嵌合抗体产物;通过HI、ELISA、免疫荧光方法验证抗体具有活性。为了在短期内累积一定的产物用于后续研究,我们选用FLP-IN定点整合表达系统,其FRT定点整合的特点使抗体能够稳定高效表达,经过细胞筛选流程,我们现已获得三株稳定表达细胞株,而且抗体的表达产量提高了3-4倍。在获得少量无血清培养的抗体纯化物后,我们用多株H5N1病毒株HI实验、及细胞体外中和实验验证嵌合抗体的广谱中和活性,以确定整个改造及表达过程中抗体的可变区活性没有受到影响。本研究选择13D4嵌合抗体进行大规模表达和纯化。为了满足下游抗体纯化简便及大规模生产的需求,我们对CHO细胞的无血清培养方法也进行了探索,包括优化无血清培养基的营养成分、滚瓶较大规模培养操作等。我们发现BSA添加物确实可以显著提高嵌合抗体的产量,尤其是3%的添加比例,可使抗体产量提高8倍,但与此同时给抗体的纯化带来干扰,应该综合考虑抗体的纯度和得率来决定最优方案;蛋白A亲和层析法一直是抗体纯化的特异有效方式,我们摸索了不同洗脱PH缓冲液,得到适合嵌合抗体纯化的洗脱PH值,最终纯度达到95%,得率在90%。针对具有代表性的四株H5N1病毒的动物保护实验结果证明,在Vietnam/1194/2005感染后的第1和3天,以及在BH Goose/Qinghai/15c/2005和Shenzhen/406H/2006感染后的第1天,13D4嵌合抗体均有100%的治疗效果。而在Indonesia/5/2005感染后的第1天和Shenzhen/406H/2006感染后的第3天,13D4嵌合抗体的治疗效果欠佳。总之,通过以上方法我们成功构建了四株高致病性禽流感H5N1人-鼠嵌合抗体,它们保留了亲本鼠抗的广谱中和活性,将降低治疗人感染疾病时的HAMA反应,为治疗性抗体在人感染H5N1领域的应用奠定了基础。
[Abstract]:Highly pathogenic avian influenza (Avian influenza AI) is a highly infectious disease caused by influenza A virus H5N1. In recent years with the continuous spread of disease, avian influenza virus not only around the poultry industry suffered huge losses, but also continue to break the species barrier caused by the incident. People infected with highly pathogenic avian influenza face the problem is the high mortality and the lack of effective drug treatment. Due to the continued rapid variation of influenza virus, the H5N1 virus has evolved in nature Mutation Strains antigenically distinct variety of Asia were popular situation, to meet the long-term strategic reserves of drug demand, H5N1 treatment of drug development must have broad spectrum.
Neutralizing antibodies is an effective means of prevention and treatment of viral diseases, but because of the bird flu virus gene mutation frequency is high, especially the hemagglutinin (HA) gene, thus neutralizing monoclonal antibodies against avian influenza virus and narrow spectrum, and only few strains, greatly limits the application of neutralizing antibodies.
Our laboratory recently constructed a hemagglutination inhibition (AI) based on H5 specific monoclonal antibody library activity (in 41 strains of H5N1 contains all 10 representative strains of these genetic clades of monoclonal antibody and the activity of HI in mice as an animal model of neutralizing activity were determined), this study selected four broad spectrum neutralizing monoclonal antibody: the 4D1,10F7,8H5,13D4 humanized reconstruction, in order to reduce the resistance in the treatment of HAMA in the reaction process, lay the foundation for the clinical application of these antibodies as soon as possible; at the same time also hope to establish a therapeutic antibody humanized research platform system, including gene expression vector transformation the construction of efficient and stable expression in mammalian cells, serum-free culture in large scale, antibody purification process.
This study first with RT-PCR from murine hybridoma cells within the variable region gene of four strains of monoclonal antibodies, combined with the constant region of human antibody IgG1, inserted into the pcDNA3.1 expression vector of chimeric antibody products obtained by HI, ELISA, instantaneous; verify the antibody immunofluorescence method with activity in the short term. In order to accumulate certain products for further study, we used site-specific integration expression system FLP-IN, characteristics of the FRT site-specific integration of the antibody to stable high expression after cell screening process, we obtained three strains of stable expression cell lines, expression and antibody production was increased by 3-4 times. In a serum-free medium to obtain antibody purified after us with H5N1 virus strain HI and in vitro experiments, broad-spectrum neutralization test verified chimeric antibody neutralizing activity, and to determine the expression of the entire transformation process of antibody variable region of no activity It has been affected.
This research chooses 13D4 chimeric antibody for large-scale expression and purification. The purification and to meet the needs of mass production, we have no culture method of serum on CHO cells were also explored, including the optimization of serum-free medium nutrient medium, large scale roller bottle culture operation. We found that the addition of BSA could significantly improve the chimeric antibody production, especially the proportion of 3%, the antibody yield increased 8 times, but at the same time the interference brought antibody purification, should consider antibody purity and yield to determine the optimal solution; protein A affinity chromatography was a specific and effective way for antibody purification, we explored different elution PH buffer, pH elution for obtained chimeric antibody purification, the final purity reached 95%, the yield is 90%.
According to the four strains of H5N1 representative of the animal protection experiment results show that in the first and third days after Vietnam/1194/2005 infection, as well as in the BH Goose/Qinghai/15c/2005 and Shenzhen/406H/2006 infection after first days, 13D4 had 100% chimeric antibody treatment. In third days after Indonesia/5/2005 infection and first days after Shenzhen/406H/2006 infection, poor 13D4 chimeric antibody treatment.
In conclusion, through the above method we successfully constructed the chimeric antibody against four strains of highly pathogenic avian influenza H5N1 human mouse, they retained broad-spectrum parental mouse neutralizing activity, will reduce the treatment of human HAMA infection during the reaction, the foundation is set for application in the foundation of human infection with the H5N1 field of therapeutic antibodies.

【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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