分子病毒学实验方法
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Chorio-allantoic membrane
Biblioteka Baidu
Air sac Yolk sac
Allantoic fluid
组织培养技术
¾ 1949年John Enders等在原代人体细胞中繁殖出脊髓灰质炎 病毒； ¾ 组织培养技术促进了病毒疫苗的研究； ¾ 1952年，R. Dulbecco利用组织培养技术发展出空斑定量病 毒的方法，首次测定脊髓灰质炎病毒和西方马脑炎病毒的侵 染性颗粒。
The Nobel Prize in Chemistry 1993 "for contributions to the developments of methods within DNA-based chemistry" "for his invention of the polymerase chain reaction (PCR) method"
Mg2+ ions: form a soluble complex with dNTP's which is essential for dNTP incorporation stimulate polymerase activity increase the Tm (melting temperature) of primer/template interaction (i.e. it serves to stabilize the duplex interaction Generally, low Mg2+ leads to low yields (or no yield) high Mg2+ leads to accumulation of nonspecific products (mispriming).
Influenza virus, negative stain
X线晶体衍射法
分子生物学方法
Polymerase Chain Reaction (PCR)
ustcwhm@ustc.edu.cn
Kary B. Mullis
*One Friday night I was driving, ……. My girlfriend, Jennifer Barnett, was asleep. I was thinking. ……. *However, shocking to me, not one of my friends or colleagues would get excited over the potential for such a process. *In September I did my first experiment. I like to try the easiest possibilities first. So one night I put human DNA and the nerve growth factor primers in a little screw-cap tube with an O-ring and a purple top. I boiled for a few minutes, cooled, added about 10 units of DNA polymerase, closed the tube and left it at 37°. It was exactly midnight on the ninth of September. *The first successful experiment happened on December 16th. I remember the date. It was the birthday of Cynthia, my former wife from Kansas City, who had encouraged me to write fiction and bore us two fine sons. I had strayed from Cynthia eventually to spend two tumultuous years with Jennifer.
血清学/免疫学方法
• 免疫沉淀法 • 中和试验 • 补体结合试验 • 免疫荧光法 • 酶联免疫吸附测定法
Hemagglutination- inhibition assay
(Differentiates between members of the same group)
RBC
RBC + Virus
Natural or recombinant Natural Recombinant Recombinant Natural Natural Recombinant Recombinant Recombinant Natural Recombinant
Source Thermus aquaticus T. aquaticus T. aquaticus Thermus flavis T. flavis Thermococcus litoralis Pyrococcus GB-D Thermus thermophilus Pyrococcus furiosus Thermotoga maritima
Kary B. Mullis La Jolla, CA, USA B:1944
The PCR Song
There was a time when to amplify DNA, You had to grow tons and tons of tiny cells. Then along came a guy named Dr. Kary Mullis, Said you can amplify in vitro just as well. Just mix your template with a buffer and some primers, Nucleotides and polymerases, too. Denaturing, annealing, and extending. Well it’s amazing what heating and cooling and heating will do. PCR, when you need to detect mutations. PCR, when you need to recombine. PCR, when you need to find out who the daddy is. PCR, when you need to solve a crime.
95 °C half-life 5'3' exo 3'5' exo Extension rate (nt/sec) RT activity Resulting ends Strand displacement M.W. (kDa)
40 min +
2.Buffers and MgCl2 in PCR reactions
PCR反应混合物
Mg Mn
2+
和 或
Taq DNA 多 聚 酶 rTth DNA 多 聚 酶 AmpErase
（UNG酶）
2+
和
dCTP dGTP dUTP dATP
引 物
1.Choice of Polymerases for PCR
DNA Polymerase Taq Amplitaq®
Amplitaq (Stoffel fragment)®
Hot Tub™ Pyrostase™ Vent™ Deep Vent™ Tth Pfu ULTma™
Properties of DNA polymerases used in PCR
Taq/ Amplitaq® Stoffel fragment 80 min Vent™ 400min Deep Vent™ 1380 min Pfu >120 min Tth 20 min + + 75 Weak 3' A >50 Weak 3' A >80 ? >95%blu nt + 94 61 ? + ? ? >95% blunt + ? 92 94 70 + 60 ? blunt >33 Yes 3' A + ? ? blunt ULTma™ >50 min
RBC + Virus + antibody to homologous virus
RBC + Virus + antibody to heterologous virus
病毒超微结构的研究方法
物理分析方法： 不同孔径的滤膜：测定病毒粒子的直径 超速离心：病毒粒子沉降特性的研究和纯化病毒 分光光度计：病毒的核酸、蛋白比率和物理学性质 电泳：从蛋白质和核酸分子水平分析病毒粒子的组成 X-射线晶体衍射技术：原子水平上测定病毒的结构 核磁共振 电子显微镜技术：病毒粒子的数量、病毒的三维外观和结构。 电子显微镜技术
病毒学研究方法
活的宿主体系 组织培养技术 血清学/免疫学方法 病毒超微结构的研究方法 分子生物学方法
活的宿主体系：植物、动物
¾ 起初的病毒学研究是建立在活的宿主体系之上的。 ¾ 动物宿主体系主要应用在生产不能体外繁殖的病毒、研究 病毒感染的病理作用、检测疫苗的安全性等。 ¾ 植物宿主体系目前应用仍较广泛
A typical reaction buffer for PCR would something like: 10mM Tris, pH 8.3 50mM KCl 1.5mM MgCl2 0.01% gelatin
The MgCl2 concentration in the final reaction mixture is usually between 0.5 to 5.0mM, and the optimum concentration is determined empirically (typically between 1.0 - 1.5mM).
¾ 20世纪初．鸡胚开始用于病毒的研究。 ¾ 1943，我国学者黄祯祥就开始利用鸡胚组织块在试管内进行 病毒传代、定量滴定及中和实验。 ¾ 1948-1955年，动物病毒的研究从利用实验动物整体过渡到 利用组织培养。小鼠、昆虫、鱼、猪、狗以及各种灵长类包括 人的Hela细胞系的体外培养相继建立。
Embryonated egg: the source of influenza vaccine today
动物细胞培养
• 开辟了定量动物病毒学的新领域以及研究病毒的复制及其与 寄主相互关系的新技术。 • 动物细胞组织培养技术的开发使脊髓灰质炎病毒的疫苗能在 体外培养的猴肾细胞中培养并大规模生产。 • 利用组培技术能较方便地诱导减毒弱株系，开发新疫苗。
Adenoviruses plaques
Poliovirus plaques
Fibroblasts in culture
Epithelial cells in culture
Cytopathic effect induced by adenoviruses
Uninfected cells
Infected cells
Measles virus - induced polykaryocytes
3.Primers
Primer design (1) Generally, primers used are 20 - 30mer in length. This provides for practical annealing temperatures (of the high temperature regimen where the thermostable polymerase is most active). (2)Primers should avoid stretches of polybase sequences (e.g. poly dG) or repeating motifs – these can hybridize with inappropriate register on the template. (3)Inverted repeat sequences should be avoided so as to prevent formation of secondary structure in the primer, which would prevent hybridization to template
病毒学研究方法
诊断技术的发展历史
20世纪60、70年代 免疫学技术-电镜-动物实验-细胞培养: 20世纪80年代 分子生物学:
HBV，HAV ，HDV HCV ，HEV
1995年 美国GBV-C/HGV ？ 1997年 日本TTV (Transfusion Transimitted Virus) ？ 最近 SEN (在一位首写名为SEN的HIV静脉吸毒者患 者血液中发现了一种新的DNA病毒) ？
Biblioteka Baidu
Air sac Yolk sac
Allantoic fluid
组织培养技术
¾ 1949年John Enders等在原代人体细胞中繁殖出脊髓灰质炎 病毒； ¾ 组织培养技术促进了病毒疫苗的研究； ¾ 1952年，R. Dulbecco利用组织培养技术发展出空斑定量病 毒的方法，首次测定脊髓灰质炎病毒和西方马脑炎病毒的侵 染性颗粒。
The Nobel Prize in Chemistry 1993 "for contributions to the developments of methods within DNA-based chemistry" "for his invention of the polymerase chain reaction (PCR) method"
Mg2+ ions: form a soluble complex with dNTP's which is essential for dNTP incorporation stimulate polymerase activity increase the Tm (melting temperature) of primer/template interaction (i.e. it serves to stabilize the duplex interaction Generally, low Mg2+ leads to low yields (or no yield) high Mg2+ leads to accumulation of nonspecific products (mispriming).
Influenza virus, negative stain
X线晶体衍射法
分子生物学方法
Polymerase Chain Reaction (PCR)
ustcwhm@ustc.edu.cn
Kary B. Mullis
*One Friday night I was driving, ……. My girlfriend, Jennifer Barnett, was asleep. I was thinking. ……. *However, shocking to me, not one of my friends or colleagues would get excited over the potential for such a process. *In September I did my first experiment. I like to try the easiest possibilities first. So one night I put human DNA and the nerve growth factor primers in a little screw-cap tube with an O-ring and a purple top. I boiled for a few minutes, cooled, added about 10 units of DNA polymerase, closed the tube and left it at 37°. It was exactly midnight on the ninth of September. *The first successful experiment happened on December 16th. I remember the date. It was the birthday of Cynthia, my former wife from Kansas City, who had encouraged me to write fiction and bore us two fine sons. I had strayed from Cynthia eventually to spend two tumultuous years with Jennifer.
血清学/免疫学方法
• 免疫沉淀法 • 中和试验 • 补体结合试验 • 免疫荧光法 • 酶联免疫吸附测定法
Hemagglutination- inhibition assay
(Differentiates between members of the same group)
RBC
RBC + Virus
Natural or recombinant Natural Recombinant Recombinant Natural Natural Recombinant Recombinant Recombinant Natural Recombinant
Source Thermus aquaticus T. aquaticus T. aquaticus Thermus flavis T. flavis Thermococcus litoralis Pyrococcus GB-D Thermus thermophilus Pyrococcus furiosus Thermotoga maritima
Kary B. Mullis La Jolla, CA, USA B:1944
The PCR Song
There was a time when to amplify DNA, You had to grow tons and tons of tiny cells. Then along came a guy named Dr. Kary Mullis, Said you can amplify in vitro just as well. Just mix your template with a buffer and some primers, Nucleotides and polymerases, too. Denaturing, annealing, and extending. Well it’s amazing what heating and cooling and heating will do. PCR, when you need to detect mutations. PCR, when you need to recombine. PCR, when you need to find out who the daddy is. PCR, when you need to solve a crime.
95 °C half-life 5'3' exo 3'5' exo Extension rate (nt/sec) RT activity Resulting ends Strand displacement M.W. (kDa)
40 min +
2.Buffers and MgCl2 in PCR reactions
PCR反应混合物
Mg Mn
2+
和 或
Taq DNA 多 聚 酶 rTth DNA 多 聚 酶 AmpErase
（UNG酶）
2+
和
dCTP dGTP dUTP dATP
引 物
1.Choice of Polymerases for PCR
DNA Polymerase Taq Amplitaq®
Amplitaq (Stoffel fragment)®
Hot Tub™ Pyrostase™ Vent™ Deep Vent™ Tth Pfu ULTma™
Properties of DNA polymerases used in PCR
Taq/ Amplitaq® Stoffel fragment 80 min Vent™ 400min Deep Vent™ 1380 min Pfu >120 min Tth 20 min + + 75 Weak 3' A >50 Weak 3' A >80 ? >95%blu nt + 94 61 ? + ? ? >95% blunt + ? 92 94 70 + 60 ? blunt >33 Yes 3' A + ? ? blunt ULTma™ >50 min
RBC + Virus + antibody to homologous virus
RBC + Virus + antibody to heterologous virus
病毒超微结构的研究方法
物理分析方法： 不同孔径的滤膜：测定病毒粒子的直径 超速离心：病毒粒子沉降特性的研究和纯化病毒 分光光度计：病毒的核酸、蛋白比率和物理学性质 电泳：从蛋白质和核酸分子水平分析病毒粒子的组成 X-射线晶体衍射技术：原子水平上测定病毒的结构 核磁共振 电子显微镜技术：病毒粒子的数量、病毒的三维外观和结构。 电子显微镜技术
病毒学研究方法
活的宿主体系 组织培养技术 血清学/免疫学方法 病毒超微结构的研究方法 分子生物学方法
活的宿主体系：植物、动物
¾ 起初的病毒学研究是建立在活的宿主体系之上的。 ¾ 动物宿主体系主要应用在生产不能体外繁殖的病毒、研究 病毒感染的病理作用、检测疫苗的安全性等。 ¾ 植物宿主体系目前应用仍较广泛
A typical reaction buffer for PCR would something like: 10mM Tris, pH 8.3 50mM KCl 1.5mM MgCl2 0.01% gelatin
The MgCl2 concentration in the final reaction mixture is usually between 0.5 to 5.0mM, and the optimum concentration is determined empirically (typically between 1.0 - 1.5mM).
¾ 20世纪初．鸡胚开始用于病毒的研究。 ¾ 1943，我国学者黄祯祥就开始利用鸡胚组织块在试管内进行 病毒传代、定量滴定及中和实验。 ¾ 1948-1955年，动物病毒的研究从利用实验动物整体过渡到 利用组织培养。小鼠、昆虫、鱼、猪、狗以及各种灵长类包括 人的Hela细胞系的体外培养相继建立。
Embryonated egg: the source of influenza vaccine today
动物细胞培养
• 开辟了定量动物病毒学的新领域以及研究病毒的复制及其与 寄主相互关系的新技术。 • 动物细胞组织培养技术的开发使脊髓灰质炎病毒的疫苗能在 体外培养的猴肾细胞中培养并大规模生产。 • 利用组培技术能较方便地诱导减毒弱株系，开发新疫苗。
Adenoviruses plaques
Poliovirus plaques
Fibroblasts in culture
Epithelial cells in culture
Cytopathic effect induced by adenoviruses
Uninfected cells
Infected cells
Measles virus - induced polykaryocytes
3.Primers
Primer design (1) Generally, primers used are 20 - 30mer in length. This provides for practical annealing temperatures (of the high temperature regimen where the thermostable polymerase is most active). (2)Primers should avoid stretches of polybase sequences (e.g. poly dG) or repeating motifs – these can hybridize with inappropriate register on the template. (3)Inverted repeat sequences should be avoided so as to prevent formation of secondary structure in the primer, which would prevent hybridization to template
病毒学研究方法
诊断技术的发展历史
20世纪60、70年代 免疫学技术-电镜-动物实验-细胞培养: 20世纪80年代 分子生物学:
HBV，HAV ，HDV HCV ，HEV
1995年 美国GBV-C/HGV ？ 1997年 日本TTV (Transfusion Transimitted Virus) ？ 最近 SEN (在一位首写名为SEN的HIV静脉吸毒者患 者血液中发现了一种新的DNA病毒) ？