小窝蛋白1对流感病毒增殖的影响及细胞培养流感病毒工艺探索
文献类型：学位论文
| 作者 | 孙李靖 |
| 学位类别 | 博士 |
| 答辩日期 | 2010-12-02 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 苏志国 ; Manfred Wirth |
| 关键词 | 小窝蛋白1 流感病毒 M2蛋白 MDCK细胞 流感疫苗 |
| 其他题名 | The influence of caveolin-1 on influenza virus propagation and research on cell culture-based influenza virus production |
| 学位专业 | 生物化工 |
| 中文摘要 | 流行性感冒（流感）严重威胁着人类健康。本论文分别以流感的治疗和疫苗生产为背景，研究分析了小窝蛋白1（caveolin-1，简写为Cav-1）对甲型流感病毒株A/PR/8/34(H1N1)增殖的影响，并对狗肾细胞（Madine-Darby canine kidney，MDCK）培养制备流感疫苗的生产工艺进行了初步探讨。第一部分着眼于流感的治疗，研究Cav-1在甲型流感病毒增殖过程中的作用。首先使用逆转录病毒介导的RNA干扰技术和瞬时转染技术改变MDCK和鼠胚纤维原（mouse embryonic fibroblasts，MEF）细胞系中Cav-1的表达水平，然后用流感病毒感染细胞，测定感染后一定时间内病毒的感染活性滴度，结果表明Cav-1在MDCK细胞系中对流感病毒的增殖既有促进作用又有抑制作用，而在MEF细胞系中对流感病毒的增殖仅表现抑制作用。其次构建M2_EGFP（增强型绿色荧光蛋白，enhanced green fluorescence protein）融合蛋白的表达质粒进行竞争实验，结果表明过量表达M2_EGFP融合蛋白能够降低流感病毒的感染活性。为了阐明CBD在Cav-1/M2结合中的作用，分别将野生型M2和CBD突变的M2蛋白表达质粒转染细胞，之后进行免疫共沉淀实验（co-immunoprecipitation，Co-IP）。结果显示，将CBD的三个核心芳香族氨基酸取代为丙氨酸或者删除CBD模体（M2氨基酸序列中47-55位氨基酸）均能削弱Cav-1/M2蛋白间的结合作用。此外，随着氨基酸长度的缩减，与Cav-1的相互作用也在逐渐减弱。这些研究证实了M2蛋白中CBD是Cav-1的结合位点。同时，观察到M2蛋白中还可能存在其他的Cav-1结合域。通过荧光显微镜观察感染及转染的细胞中Cav-1和M2蛋白的分布，发现两者主要共定位在浆膜和核周区域，删除了CBD或者完全截去羧基尾域的M2蛋白在细胞中主要均匀分布在细胞质中，表明CBD或包含兼性螺旋的区域在新合成的M2蛋白的定位和转运过程中起着重要作用。M2/Cav-1的相互作用为抗流感病毒感染提供了一条潜在的治疗途径。第二部分侧重研究流感疫苗的生产，旨在用MDCK细胞取代鸡胚培养流感病毒，开发新型安全高效的流感疫苗生产工艺。首先研究比较了六株鸡胚适应型流感疫苗生产株在MDCK细胞中的增殖能力，发现季节性甲型流感病毒株IVR-148的增殖能力最强，而甲3型流感病毒株的增殖量最少。其次从病毒培养的影响因素入手，探索了胰酶、病毒感染复数、胎牛血清、牛血清白蛋白、人血清白蛋白，以及温度对流感病毒株IVR-148增殖的影响。在优化的实验条件下，IVR-148病毒株在50ml转管中培养的MDCK细胞中增殖可以达到1:1280的血凝滴度，与在鸡胚中增殖获得的血凝滴度相当。在此基础上进行了10L反应器规模的中试放大，IVR-148病毒株的血凝滴度最高达到1:320，培养工艺条件和病毒株均有待进一步优化。 |
| 英文摘要 | Influenza (flu) has been one of the most serious life-threatening diseases to humankind. Based on influenza therapy and vaccine production respectively, present thesis investigated the influence of caveolin-1 (Cav-1) on the propagation of influenza A virus A/PR/8/34(H1N1). Moreover, Madine-Darby canine kidney (MDCK) cell culture-based influenza vaccine production technique was explored. The first part of this thesis focuses on therapy of influenza. The role of Cav-1 in influenza A virus multiplicity cycle was investigated. First of all, retrovirus-mediated RNA interference and transient transfection techniques were applied to knock down or overexpress Cav-1 in MDCK or MEF (mouse embryonic fibroblasts) cell lines followed by infection with influenza virus, the infectivity titers of influenza virus were determined in a time course pattern, Cav-1 was indicated both promotion and inhibition effects on the multiplication of influenza virus in MDCK cells, while only inhibition effect was verified in MEF cells. Plasmids expressing M2_EGFP (enhanced green fluorescence protein) fusion proteins were constructed for competition experiments, and decreased infectivity of influenza virus in those cells overexpressing M2_EGFP fusion proteins was detected. Co-immunoprecipitation (Co-IP) experiments encompassing wild-type M2 as well as CBD mutants transfected via expression vectors were performed to clarify the role of CBD in the Cav-1/M2 interaction. Replacement of the aromatic core residues with alanines or deletion of the CBD region (positions 47-55 in the M2 amino acid sequence) impaired the Cav-1/M2 binding in these experiments. Additionally, M2 proteins with C-terminal truncations in the cytoplasmic tail attenuated the interaction in a gradual pattern in parallel with the shortening of the M2 tail. These investigations confirmed the function of the CBD domain of the M2 protein that as a binding site with Cav-1. Meanwhile, the existence of another Cav-1 binding domain in M2 is suggested. Inspection of the distribution of Cav-1 and M2 in infected and transfected cells using fluorescence microscopy revealed that both proteins mainly co-localized in the plasma membrane and perinuclear areas. Deletion of the CBD or complete truncation of the cytoplasmic tail results in redistribution of M2 in the cytoplasm, indicating a role of the CBD or encompassing helical region in localization and transport of newly synthesized M2. The observed interplay of M2 and Cav-1 proteins provides a potential therapeutic way for intervention with influenza virus infection. The second part of present thesis focuses on influenza vaccine production, intending to use MDCK cells instead of embryonated eggs for influenza virus propagation, and to develop a new safe and highly efficient influenza vaccine production technique. Firstly, the propagation abilities of six egg-based influenza strains used for vaccine production were investigated in MDCK cells. It was found that seasonal influenza A virus strain IVR-148 had the most efficient proliferation rate in MDCK cells, while an influenza A H3 subtype strain propagated the least. Secondly, the effects of environmental factors, including trypsin, multiplicity of infection (MOI), fetal bovine serum (FBS), bovine serum albumin (BSA), human serum albumin (HSA) and temperature on the multiplication of influenza virus stain IVR-148 were investigated. Under optimal experiment conditions, the HA titer of IVR-148 propagated in 50 ml-tube with MDCK cells was 1:1280, which was the same as in eggs. Based on these findings, influenza viruses were cultivated in a 10L bioreactor for scale-up production. The highest HA titer that IVR-148 virus could reach is 1:320. The culture tech needs to be further optimized with respect to the performance criterion and appropriate virus strain. |
| 语种 | 中文 |
| 公开日期 | 2013-09-22 |
| 页码 | 159 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1631]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 孙李靖. 小窝蛋白1对流感病毒增殖的影响及细胞培养流感病毒工艺探索[D]. 中国科学院研究生院. 2010. |
入库方式： OAI收割
来源：过程工程研究所
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