Influenza virus, negative stain
X线晶体衍射法
分子生物学方法
Polymerase Chain Reaction (PCR)
ustcwhm@
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.
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)®
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
Chorio-allaac 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。
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).
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"
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
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
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).
Fibroblasts in culture
Epithelial cells in culture
Cytopathic effect induced by adenoviruses
Uninfected cells
Infected cells
Measles virus - induced polykaryocytes
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
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
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) ？
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
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。