乙型流感病毒Vero細(xì)胞適應(yīng)株的選育及其基因研究
發(fā)布時(shí)間：2019-03-17 12:43
【摘要】： 流感病毒(influenza virus)是一種能引起急性呼吸道疾病的病毒,具有高度傳染性,主要經(jīng)空氣飛沫傳播,因此它能在短期內(nèi)突然發(fā)生,迅速蔓延,造成不同程度的流行。流行性感冒(流感)病毒在世界范圍內(nèi)引起人畜的發(fā)病和死亡。目前對流感的防治主要是接種流感疫苗。乙型流感病毒疫苗是其重要的組成部分。傳統(tǒng)制備疫苗的方法是使用雞胚生產(chǎn),存在很多缺陷和局限性,應(yīng)用哺乳動(dòng)物細(xì)胞生產(chǎn)流感疫苗是個(gè)更好的選擇。Vero細(xì)胞是WHO積極推薦用于疫苗生產(chǎn)的細(xì)胞基質(zhì)。在乙型流感疫苗的研制中,乙型流感病毒感染很難適應(yīng)Vero細(xì)胞,并且很難得到持續(xù)的高產(chǎn)。在大量臨床樣本的基礎(chǔ)上,本論文成功篩選到一株乙型流感病毒Vero細(xì)胞適應(yīng)株,且能達(dá)到高產(chǎn),穩(wěn)定的目的。將篩選到的毒株進(jìn)行一系列的抗原性分析和基因性分析,這些研究為Vero細(xì)胞乙型流感疫苗的開發(fā)提供前提。該毒株可以作為親本株,采用遺傳重配或反向遺傳學(xué)技術(shù)將其與流行毒株重配,制備Vero細(xì)胞乙型流感疫苗生產(chǎn)毒種。 第一部分乙型流感病毒在Vero細(xì)胞上的適應(yīng)株的選育 本實(shí)驗(yàn)在乙型流感病毒很難在Vero細(xì)胞上適應(yīng)的情況下,首先探索病毒的培養(yǎng)條件。確定病毒培養(yǎng)液中的TPCK-胰酶終濃度1μg/ml,NaHCO3終濃度198μg/ml。和采用MEM作為基礎(chǔ)培養(yǎng)基同時(shí)不添加谷氨酰胺等條件。在30株臨床樣本毒株中最終選育出一株乙型流感病毒Vero細(xì)胞高產(chǎn)適應(yīng)株,連續(xù)傳代21個(gè)代次,結(jié)果穩(wěn)定。同時(shí)進(jìn)行相關(guān)生物學(xué)檢測。 第二部分乙型流感病毒Vero細(xì)胞適應(yīng)株B/Yunnan/02/2005(B)的基因分析 本實(shí)驗(yàn)是在成功獲得一株乙型流感病毒Vero細(xì)胞高產(chǎn)適應(yīng)株的基礎(chǔ)上。重點(diǎn)對該毒株的基因性進(jìn)行研究。通過RT-PCR的方法獲得該毒株的四個(gè)基因片段。通過對HA1,M,NA,NP進(jìn)行BLAST分析,認(rèn)識適應(yīng)株的基因背景。同時(shí)將HA1基因作為研究的重點(diǎn),與國際流行株進(jìn)行比對,計(jì)算出遺傳距離,構(gòu)建HA1基因進(jìn)化樹。將該毒株和WHO從2004年到2010年推薦疫苗株進(jìn)行比較和分析,找到氨基酸替換位點(diǎn)。
[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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R373
本文編號：2442311
[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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R373
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，本文編號：2442311
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