H7N9亚型流感病毒样颗粒免疫原制备与精制免疫球蛋白研究

发布时间：2018-01-07 20:33

本文关键词：H7N9亚型流感病毒样颗粒免疫原制备与精制免疫球蛋白研究　出处：《中国人民解放军军事医学科学院》2017年博士论文　论文类型：学位论文

【摘要】：H7N9亚型流感病毒是一种新发流感病毒,于2013年3月在上海和安徽两地首次发现,截止到2017年2月22日,全球共报道1230例H7N9亚型流感病毒感染人的病例,其中428例死亡,死亡率达到34.80%。目前,多个国家已经着手开展H7N9亚型流感病毒病诊断、防治等相关产品的研发工作。对于高风险人群,接种疫苗是一种有效的预防手段;对于易感人群和感染患者,被动免疫则是一种有效的紧急预防和治疗手段。因此,有必要研制安全、有效、经济实用的流感疫苗和治疗抗体。流感病毒的致病性主要受到病毒的分子特性和宿主的种属差异影响,构建流感病毒动物感染模型,开展病毒的致病性分子机制研究,将有助于我们有针对性地对该病进行免疫预防和治疗研究。Palladino等用中和单抗治疗免疫缺陷的A型流感病毒感染小鼠,显示出良好的治疗效果,提示抗体疗法可能成为未来对抗H7N9亚型流感有效的手段之一。人源抗体是最理想的治疗抗体,但是短期难以获得大量的人源血清,因此异源抗体(动物源免疫球蛋白)成为现阶段必然的抗体治疗替代手段之一。在各种动物中,马抗血清制品生产和应用已有100多年的历史,具有易于大规模制备、特异性好、制备周期相对较短等优点,在应对传统疾病和新发/突发传染病中发挥了巨大作用。本研究以研制马抗H7N9亚型流感精制免疫球蛋白为研究目标,首先建立了H7N9亚型流感病毒小鼠感染模型,为H7N9流感治疗性抗体研究提供了评价平台;其次,利用昆虫细胞—杆状病毒表达系统,包装出含有A/Shanghai/2/2013(H7N9)毒株HA、NA和M1蛋白的病毒样颗粒(virus-like particles,VLPs),并对VLPs进行体外生物学活性检测和小鼠体内免疫原性研究;最后,以VLPs作为免疫原免疫马匹,通过优化生产工艺对马高免血清进一步精制,获得了马抗H7N9亚型流感病毒精制免疫球蛋白F(ab′)2,并评价其预防和治疗效果。1、H7N9亚型流感病毒小鼠感染模型的构建及致病性分子基础研究选取BALB/c小鼠作为实验动物模型,将A/Shanghai/2/2013(H7N9)毒株通过鼠肺连续传代获得鼠适应株P10M1、P10M2和P10M3。通过小鼠半数致死量、病死率、病毒复制效率、组织嗜性等评价指标,成功建立了H7N9亚型流感病毒小鼠感染模型,为H7N9亚型流感治疗药物和疫苗预防效果评价建立了平台。基因组序列分析发现,PA、HA、NA、M和NP基因上的若干个位点发生变异,推测发现其中PA-T97I可能是H7N9亚型流感病毒对哺乳动物致病性增强的分子基础之一。2、H7N9亚型流感病毒样颗粒制备利用昆虫细胞——杆状病毒表达系统,构建了表达A/Shanghai/2/2013(H7N9)毒株HA、NA和M1的重组杆状病毒,经PCR、间接免疫荧光和Western blot鉴定,目的蛋白获得成功表达。将重组杆状病毒感染昆虫细胞后成功组装成H7N9亚型流感病毒样颗粒在透射电镜下可观察到球形、直径约100nm、表面有典型纤突的流感VLPs,血凝效价为1:27~1:28。将VLPs通过肌肉注射或滴鼻方式,免疫BALB/c小鼠10μg VLPs/只,二免后两周,在H7N9亚型流感病毒小鼠适应株10MLD50剂量的攻击下,可以对小鼠提供100%的免疫保护。因此,所制备的H7N9亚型流感VLPs具有很好的免疫原性,为H7N9亚型流感免疫球蛋白的免疫原制备奠定了基础。3、H7N9亚型流感病毒精制免疫球蛋白研究以VLPs制备免疫原免疫马匹,五免后获得HI抗体效价为1:10240的高免血清。利用已构建的H7N9亚型流感病毒小鼠感染模型,用10个MLD50采用滴鼻方式进行攻毒,攻毒前36h、攻毒后24h,各组小鼠分别腹腔注射0.2ml高免血清,小鼠的存活率均达100%,而对照组全部死亡。由此证明,制备的马抗H7N9亚型流感病毒高免血清对该亚型流感病毒感染具有良好的紧急预防和治疗效果。在传统马抗血清纯化工艺的基础上,对制备工艺进行了改进和优化,采用酶切纯化工艺去除抗体的Fc片段,亲和柱层析方法纯化免疫球蛋白F(ab′)2。经SDS-PAGE、薄层扫描,结果表明,所获得的精制免疫球蛋白F(ab′)2的纯度在93%以上。采用固定病毒—稀释免疫球蛋白方法,测得的中和抗体效价为1:5120。利用已构建的H7N9亚型流感病毒小鼠感染模型,用10个MLD50采用滴鼻方式进行攻毒,攻毒前4h、攻毒感染后8h,0.2mg给药剂量即可100%保护小鼠;攻毒24h后,0.4mg给药剂量的小鼠存活率为75%。小鼠实验证明,制备的精制免疫球蛋白F(ab′)2对H7N9亚型流感有较好的预防和治疗效果。综上所述,本研究通过构建感染模型发现了H7N9对哺乳动物致病的关键分子标记。进而,利用所构建的H7N9亚型流感病毒小鼠感染模型证实H7N9亚型流感VLPs具有良好的免疫保护效果,而且马抗H7N9亚型流感VLPs精制免疫球蛋白F(ab′)2在预防和治疗H7N9亚型流感中表现出潜在的应用价值,为H7N9亚型流感治疗药物和疫苗的研制提供了科技支撑。
[Abstract]:H7N9 subtype influenza virus is a new influenza virus, first discovered in March 2013 in Shanghai and Anhui, by the end of February 22, 2017, the global total reported 1230 cases of influenza a H7N9 virus infection, including 428 deaths, the mortality rate reached 34.80%. at present, many countries have embarked on the H7N9 influenza virus diagnosis disease prevention and other related product development work. For the high risk population, vaccination is an effective means of prevention; for susceptibles and infected patients, passive immunization is an effective prevention and emergency treatment. Therefore, it is necessary to develop safe, effective, economical and practical treatment of influenza vaccine and antibody the pathogenicity of influenza virus is mainly affected by the molecular characteristics of virus and host species differences, construction of influenza virus infected animal model of pathogenic molecular mechanism of virus will be carried out. Help us for the disease prevention and treatment of immune.Palladino A influenza virus neutralizing monoclonal antibodies for the treatment of immunodeficiency infected mice, showed good therapeutic effect, suggesting that antibody therapy could be effective against future influenza H7N9 in one of the human antibody is the most ideal therapeutic antibodies but the short term is difficult to obtain a large number of people from the serum, so the heterologous antibody (animal IgG) antibody treatment at this stage will become one of the alternative means. In a variety of animal, horse serum products production and use of more than 100 years of history, it is easy to large-scale preparation, good specificity, preparation the advantages of the relatively short period, in response to the traditional disease and new onset / infectious disease has played an important role in this study. Based on the development of horse anti influenza H7N9 purified immunoglobulin as the research object, the first We first establish a H7N9 influenza virus infected mice model, provides a platform for the evaluation of therapeutic antibodies of H7N9 influenza; secondly, the expression system in insect cells, baculovirus, packaging containing A/Shanghai/2/2013 (H7N9) HA strain, virus like particles of NA and M1 protein (virus-like particles, VLPs), and study the original in vitro biological activity test and mice immune to VLPs; finally, using VLPs as immunogen by optimizing the production process of horses, horse serum was obtained for further refining, horse anti H7N9 virus purified immunoglobulin influenza virus subtype F (AB) 2, and evaluate the effect of prevention and treatment of.1. H7N9 subtype of influenza virus infection in mice model and study the pathogenic molecular basis of selected BALB/c mice as experimental animal model, A/Shanghai/2/2013 (H7N9) strains by rat lung even to obtain mouse adapted strain P10M generation http 1, P10M2 and P10M3. in mice by median lethal dose, mortality, viral replication efficiency, tissue tropism and evaluation index, successfully established the H7N9 subtype of influenza virus infected mice model was established to evaluate the preventive effect of H7N9 platform influenza therapeutics and vaccines. Genomic sequence analysis showed that PA, HA, NA and several sites of M and NP gene mutation, which may be speculated that the discovery of PA-T97I H7N9 subtype influenza virus on the molecular basis of mammalian pathogenicity enhanced.2, H7N9 subtype influenza virus like particles prepared by insect cells, baculovirus expression system, constructed the expression of A/Shanghai/2/2013 (H7N9) HA strain, recombinant baculovirus, NA and M1 by PCR, indirect immunofluorescence and Western blot identification, the target protein was successfully expressed. The recombinant baculovirus infected insect cells successfully assembled H7N9 subtype influenza virus like. The particles could be observed under TEM spherical, diameter of about 100nm, the surface of the typical spike of influenza VLPs, hemagglutination titer of 1:27~1:28. VLPs by intramuscular injection or nasal drops, BALB/c mice were immunized with 10 g VLPs/, two, two weeks after immunization, in H7N9 subtype influenza virus strain 10MLD50 dose in mice the attack, can provide protective immunity in mice 100%. Therefore, the preparation of the H7N9 subtype of influenza VLPs has good immunogenicity, immune system H7N9 influenza immune globulin preparation has laid the foundation of.3, H7N9 subtype influenza virus purified immunoglobulin on VLPs system preparation of immunized horses, five free after HI antibody titer of hyperimmune serum 1:10240. Using the H7N9 subtype of influenza virus infection in mice has been constructed model, were challenged with 10 MLD50 with nasal drops, before attacking poison 36h, after infection of 24h mice respectively by intraperitoneal injection of 0. 2ml hyperimmune serum, the survival rate of mice was 100%, while the control group all died. This proves that the preparation of horse anti influenza virus subtype H7N9 hyperimmune serum with emergency good prevention and treatment effects of the infection of influenza virus. Based on the traditional horse anti serum purification technology, improved and the optimization of preparation process, using enzyme digestion and purification to remove the antibody Fc fragment, affinity chromatography purification of immunoglobulin F (AB) 2. by SDS-PAGE, TLC scanning, the results show that the purified immunoglobulins obtained by F (AB) 2 purity was above 93%. The fixed virus immunoglobulin - dilution method, the neutralizing antibody titers measured for 1:5120. by H7N9 subtype of influenza virus infection in mice has been constructed model, were challenged with 10 MLD50 with nasal drops, before attacking poison 4H infection after 8h and 0.2mg administration can protect small dose 100% Rat; attack 24h, 0.4mg dosage, the survival rate of mice was demonstrated in 75%. mice, purified preparation of immunoglobulin F (AB) has good preventive and therapeutic effect of 2 H7N9 influenza. In summary, this study builds the infection model of H7N9 was found on key molecules in mammals pathogenic markers. Furthermore, the use of H7N9 subtype influenza virus infection model of mice confirmed H7N9 influenza VLPs has good immune protection effect, and horse anti influenza H7N9 VLPs purified immunoglobulin F (AB) 2 in the prevention and treatment of influenza H7N9 showed potential application value and to provide scientific support for the development of influenza H7N9 therapeutics and vaccines.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

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