乙型流感病毒Vero细胞适应株的选育及其基因研究

发布时间：2019-03-17 12:43

【摘要】： 流感病毒(influenza virus)是一种能引起急性呼吸道疾病的病毒,具有高度传染性,主要经空气飞沫传播,因此它能在短期内突然发生,迅速蔓延,造成不同程度的流行。流行性感冒(流感)病毒在世界范围内引起人畜的发病和死亡。目前对流感的防治主要是接种流感疫苗。乙型流感病毒疫苗是其重要的组成部分。传统制备疫苗的方法是使用鸡胚生产,存在很多缺陷和局限性,应用哺乳动物细胞生产流感疫苗是个更好的选择。Vero细胞是WHO积极推荐用于疫苗生产的细胞基质。在乙型流感疫苗的研制中,乙型流感病毒感染很难适应Vero细胞,并且很难得到持续的高产。在大量临床样本的基础上,本论文成功筛选到一株乙型流感病毒Vero细胞适应株,且能达到高产,稳定的目的。将筛选到的毒株进行一系列的抗原性分析和基因性分析,这些研究为Vero细胞乙型流感疫苗的开发提供前提。该毒株可以作为亲本株,采用遗传重配或反向遗传学技术将其与流行毒株重配,制备Vero细胞乙型流感疫苗生产毒种。第一部分乙型流感病毒在Vero细胞上的适应株的选育本实验在乙型流感病毒很难在Vero细胞上适应的情况下,首先探索病毒的培养条件。确定病毒培养液中的TPCK-胰酶终浓度1μg/ml,NaHCO3终浓度198μg/ml。和采用MEM作为基础培养基同时不添加谷氨酰胺等条件。在30株临床样本毒株中最终选育出一株乙型流感病毒Vero细胞高产适应株,连续传代21个代次,结果稳定。同时进行相关生物学检测。第二部分乙型流感病毒Vero细胞适应株B/Yunnan/02/2005(B)的基因分析本实验是在成功获得一株乙型流感病毒Vero细胞高产适应株的基础上。重点对该毒株的基因性进行研究。通过RT-PCR的方法获得该毒株的四个基因片段。通过对HA1,M,NA,NP进行BLAST分析,认识适应株的基因背景。同时将HA1基因作为研究的重点,与国际流行株进行比对,计算出遗传距离,构建HA1基因进化树。将该毒株和WHO从2004年到2010年推荐疫苗株进行比较和分析,找到氨基酸替换位点。
[Abstract]:Influenza virus (influenza virus) is a kind of virus which can cause acute respiratory diseases. It is highly contagious and mainly transmitted by airborne droplets. Therefore, it can suddenly occur in a short period of time and spread rapidly, resulting in different degrees of epidemics. Influenza (influenza) viruses cause the onset and death of humans and animals worldwide. At present, influenza vaccination is the main prevention and treatment of influenza. Influenza B vaccine is an important part of the vaccine. The traditional method of vaccine preparation is chicken embryo production, which has many defects and limitations. It is a better choice to produce influenza vaccine with mammalian cells. Vero cells are the cell matrix recommended by WHO for vaccine production. In the development of influenza B vaccine, influenza B virus infection is difficult to adapt to Vero cells, and it is difficult to obtain sustained high yield. On the basis of a large number of clinical samples, a strain of influenza B virus Vero cell adaptation strain was successfully screened in this paper, which can achieve the goal of high yield and stability. A series of antigenicity analysis and genetic analysis were carried out, which provided a prerequisite for the development of influenza B vaccine on Vero cells. The strain could be used as a parent strain to produce Vero cell influenza B vaccine by genetic recombination or reverse genetic technique. Part one selection of strain adapted to influenza B virus on Vero cells in this experiment the culture conditions of influenza B virus were first explored when it was difficult for influenza B virus to adapt to Vero cells. Determination of the final concentration of TPCK- trypsin in virus culture medium 1 渭 g / ml,NaHCO3 final concentration 198 渭 g / ml. MEM was used as the basic medium and glutamine was not added to the medium at the same time. A high-yield strain of influenza B virus (Vero) was selected from 30 clinical strains. The strain was passed for 21 generations and the result was stable. At the same time, related biological tests were carried out. Part two: gene analysis of influenza B virus Vero cell adaptation strain B/Yunnan/02/2005 (B). This experiment is based on the successful production of a high-yield strain of influenza B virus Vero cell. The genetic characteristics of the strain were mainly studied. Four gene fragments of the strain were obtained by RT-PCR. Through BLAST analysis of HA1,M,NA,NP, the genetic background of adaptive strain was recognized. At the same time, the HA1 gene was taken as the focus of the research, compared with the international popular strains, the genetic distance was calculated, and the evolutionary tree of HA1 gene was constructed. The amino acid substitution sites were found by comparison and analysis between the strain and the vaccine strain recommended by WHO from 2004 to 2010.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373

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