基于MDCK细胞培养的流感疫苗研究

发布时间：2019-01-27 06:49

【摘要】：流行性感冒是人类至今不能有效控制的传染病之一，一直是威胁全世界人类安全的疾病。虽然有抗病毒药物，但可能会诱导耐药株，不适合大范围长时间来应对流感病毒。疫苗是预防和控制流感的重要手段。除了传统的鸡胚制备流感疫苗，传代细胞系如MDCK细胞、Vero细胞、RER.C6细胞等也被用作制备流感疫苗。MDCK细胞具有易培养、对多种不同亚型的流感病毒株敏感、病毒用MDCK细胞培养的产量高等特点，是作为流感疫苗生产的理想细胞系。本文建立了适用于生产的MDCK细胞三级种子库，并使用该种子库开展对于MDCK细胞的生长情况的研究。通过对不同血清比较，葡萄糖浓度调整，，确定了在培养瓶中MDCK细胞最佳贴壁生长条件。在此基础上，进一步研究MDCK细胞在微载体表面反应器系统中贴壁生长情况。MDCK细胞以微载体贴壁生长，在初始接种细胞密度为2.0×105cells/mL，在不更换培养基的情况下，经过72小时，细胞密度最高能达到（13.77±0.69）×105cells/mL，细胞数量扩增了6.89倍。另外，对MDCK细胞培养流感病毒的条件进行优化。当细胞培养至72小时，按照M.O.I=0.01接种流感病毒，以含2.5μg/mL TPCK-胰酶的MEM培养基培养流感病毒。在48小时后，培养基上清中能够获得的流感病毒血凝滴度为9log2HA/50μL。为今后建立以生物反应器微载体系统大规模培养流感疫苗技术平台奠定了基础。
[Abstract]:Influenza is one of the infectious diseases which can not be effectively controlled by human beings, and it has been a threat to human security all over the world. Although antiviral drugs are available, resistant strains may be induced and are not suitable for a wide range of long-term influenza viruses. Vaccine is an important means to prevent and control influenza. In addition to the traditional chicken embryo flu vaccine, passage cell lines such as MDCK cells, Vero cells and RER.C6 cells are also used to prepare influenza vaccines. MDCK cells are easy to culture and sensitive to a variety of different subtypes of influenza viruses. The high yield of MDCK cells is the ideal cell line for influenza vaccine production. In this paper, a three-stage seed bank of MDCK cells suitable for production was established, and the growth of MDCK cells was studied by using the seed bank. By comparing different serum and adjusting the concentration of glucose, the optimal conditions of MDCK cell adhesion in culture flask were determined. On this basis, the adherent growth of MDCK cells in the microcarrier surface reactor system was further studied. MDCK cells grew on microcarriers, and the initial inoculation cell density was 2.0 脳 105 cells / mL, without changing the culture medium. After 72 hours, the maximum cell density was (13.77 卤0.69) 脳 10 ~ 5 cells / mL, and the cell number was 6.89 times. In addition, the conditions of MDCK cell culture of influenza virus were optimized. When the cells were cultured for 72 hours, influenza virus was inoculated according to M.O.I=0.01 and cultured on MEM medium containing 2.5 渭 g/mL TPCK- trypsin. After 48 hours, the hemagglutination titer of influenza virus was 9log2HA/50 渭 L. It lays a foundation for the establishment of a bioreactor microcarrier system for the large-scale cultivation of influenza vaccine technology platform in the future.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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