病毒空斑纯化实验protocal
病毒的分离纯化
病毒的分离纯化
我设计了一套方案，请大家批评指正：
1. 新鲜病叶200g，-70℃冰冻30分钟，以2倍体积0.2mol/L磷酸缓冲液（PH7.5，含0.1%巯基乙醇,2%TritionX-100,0.01mol/LEDTA）。
组织捣碎机捣碎，双层纱布过滤。
2. 滤液中加入等体积的氯仿，冰浴激烈搅拌15分钟，充分乳化后2000 g离心15分钟
3. 上清液中加入6%聚乙二醇-6000，3%Nacl,0.5%TritionX-100（W/V）。
冰浴搅拌，4℃下过夜，次日取出10000g离心20分钟。
4. 沉淀悬浮于0.02mol/L磷酸缓冲液（PH7.2，2%TritionX-100, 0.1%巯基乙醇，1mol/L尿素），2小时后，2000g离心10分钟。
5. 上清液中加入6%聚乙二醇-6000，3%Nacl,0.5%TritionX-100（W/V）。
冰浴搅拌4小时，10000g离心30分钟。
6. 沉淀悬浮于0.02mol/L磷酸缓冲液（PH
7.2，2%TritionX-100,0.01mol/LEDTA，1mol/L尿素），4℃下4小时后，10000g离心10分钟。
7. 上清用10%～40%蔗糖密度梯度离心(30000r/min,1.5h)，收集病毒带，用0.02mol/L磷酸缓冲液（PH7.2，2%TritionX-100, 0.1%巯基乙醇，1mol/L尿素）稀释，123000g离心2小时。
8.沉淀用0.02mol/L磷酸缓冲液（PH7.2，2%TritionX-100, 0.1%巯基乙醇，1mol/L尿素）悬浮，5000g离心15分钟，上清即为提纯病毒制品。
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病毒的纯化与保存
连续与不连续PAGE
PAGE按照缓冲液的pH值和凝胶孔径的差 异及有无浓缩效应分为连续系统和不连 续系统两大类,
病毒纯化的一般原则熟悉
1、释放病毒到胞外 1.1 容易释放病毒 培养液/尿囊液
1.2 不容易释放病毒 细胞破碎 2、去除细胞碎块
低速离心,以2000～6000r/min离心30～ 40分钟,可除去90％以上的细胞碎片和杂质
3、病毒悬液浓缩
细胞破碎方法熟悉
超声破碎：密集的小气泡迅速炸裂,破坏细胞 高压匀浆：机械切割力 高速珠磨：玻璃小珠、石英砂、氧化铝等研磨剂 酶溶法：溶菌酶、蛋白酶、葡聚糖酶 化学渗透：渗透压改变使细胞破裂 反复冻融：形成冰晶,使细胞膨胀破裂
每个蛋白标准的分子量对数对它的相对迁 移率作图得标准曲线,量出未知蛋白的迁移 率即可测出其分子量,这样的标难曲线只对 同一块凝胶上的样品的分子量测定才具有 可靠性，
当蛋白质的分子量在15,000－200,000之间时,样品 的迁移率与其分子量的对数呈线性关系,
符合如下方程式：Lg MW =－b m R + K 其中，MW 为蛋白质的分子量，m R 为相对迁移 率，b为斜率，K为截距，当条件一定时，b与K均 为常数
WesternBlot 步骤
3、磷酸缓冲液漂洗,进行膜的脱色 4、膜的封闭
封闭是用高浓度的无关蛋白质封闭膜上未被占据
的表面,使抗体仅仅只能跟特异的蛋白质结合而
不是和膜结合，常用的封闭液有牛血清白蛋白 BSA，脱脂奶粉等，一般用脱脂奶粉， 将膜浸没于封闭液中缓慢摇荡一小时。
M13噬菌体空斑实验
M13噬菌体空斑实验一、实验目的：1、了解M13噬菌体载体的一般结构、性质及感染特征。
2、学习并掌握M13噬菌体载体的空斑纯化技术。
二、实验原理：一个M13噬菌体的病毒颗粒感染一个细菌后形成一个噬菌斑。
从感染细菌释放出来的子代病毒颗粒感染临近细菌，后者又可释放下一代病毒颗粒；如果细菌在半固体培养基上生长，自带病毒颗粒的扩散受到一定限制，诸如M13等丝状噬菌体并不裂解细菌，但可以使细菌生长速度降低至原来的1/2，在上层琼脂中生长的菌台的背景下，生长缓慢细菌形成了一个不断扩大到肉眼可见的噬菌斑。
三、材料与试剂：1、M13噬菌体2、ER2738菌种3、LB培养基四、实验步骤1.1、取5ml LB液体培养基于10ml的试管中，加入5ul 四环素（20mg/ml），将大肠杆菌接入，37℃震荡（80-100rpm）培养过夜（12h）2.将2ml的ER2738培养液转入20ml的LB培养基中（1:10稀释），继续37℃震荡（80-100rpm）培养4.5h（不宜过快，否则，细菌性毛被破坏，噬菌体无法导入），即成为感受态细胞3.培养细菌时将水浴锅打开，43℃预热，并将LB 平板置于37℃预热备用4.用装有LB培养基的EP管10倍稀释噬菌体，理想的稀释范围：扩增过的噬菌体108-1010倍稀释，未扩增过的104-105倍稀释5.将顶层琼脂用微波炉或者电磁炉沸水融化，置于水浴锅中保温待用6.当大肠杆菌达到对数成长期，取大肠杆菌ER2738感受态细胞200ul于EP管中7.加入10ul-100 ul（或者一定体积）一定稀释度的噬菌体溶液，快速混合，室温孵育1-5min8.将感染过的大肠杆菌的培养液倒入43℃顶层琼脂中，用vortex迅速混匀，立刻在预热的LB培养基上倒平板，迅速摇晃平板使顶层琼脂均匀展开并铺满表面9.室温静置5min左右使平板冷却凝固，将培养皿倒置37℃培养过夜10.第二天观察平板，对培养皿中的噬菌斑进行计数，计算约含有10-100个数量级的平板噬菌斑的具体数目（y），计算噬菌体效价（pfu/ml）噬菌体效价=y*10x+2puf/ml分子生物学作业1、什么是细菌的转化？2、在哪些生物中，RNA是唯一的遗传物质？3、与单个氨基酸对应的碱基序列叫什么？4、DNA的复制是全保留还是半保留的？5、在半保留复制中，在第一、第二和第三轮复制中，分别有多少含有一条亲代链和一条子代链的双连DNA分子？。
病毒空斑纯化
病毒空斑纯化病毒蚀斑如同噬菌体的噬斑，一个蚀斑为一个病毒体的繁殖后代品系。
在细胞培养中做蚀斑是一种较精确的测定病毒感染的方法，将病毒各稀释度种入单层细胞瓶，吸附1h，在单层细胞上覆以营养琼脂培养基，病毒在细胞中繁殖使细胞死亡。
但由于琼脂的限制只能感染临近的细胞，形成“蚀斑”的退化细胞区，经中性红活细胞染料着色后，活细胞显红色，而蚀斑区细胞已退化步着色，形成不染色区域。
凡是能在细胞培养物中产生CPE的病毒都可以采用蚀斑技术来分离和测定。
蚀斑技术在测定病毒感染时是很好的定量方法，也是测定干扰素和抗体中和病毒繁殖能力的一种非常敏感的方法。
此外还可以用来纯化病毒，纯化时挑选合适的空斑进行传代即可。
若目的是要提高病毒能力，应选大空斑，若是为了减毒以制备疫苗应挑选小空斑，但每瓶空斑数不得超过20-40个。
试验时，培养瓶应翻转培养，以防止水滴在琼脂面流动，以免各个蚀斑交叉融合，中性红应在蚀斑出现前加入，在暗处孵育，以防止染料抑制病毒和破坏细胞。
对那些生长缓慢的病毒蚀斑，中性红则不是加在最初的覆盖层里，而是加在经过几天培养后的最后一层覆盖层上，可使培养物维持更久，甚至有的中间还得补加营养琼脂覆盖层1-2次。
蚀斑使用的琼脂往往有少量抑制物而影响蚀斑的产生，应将琼脂用单蒸水浸泡过夜，再用单蒸水冲洗2-3次，而后用去离子水冲洗1-2次后，用Hanks液配制成3%浓度，煮沸1h 除菌备用。
有人在试验时补加25mmol的MgCl2或CaCl2及1mmol的半胱氨酸，可加强肠道病毒形成蚀斑。
而MgCl2的加强作用最强。
流感病毒纯化（蔗糖梯度法）概论转头选择密度梯度选用一、概论在密度梯度离心中单一样品组份的分离是借助于混合样品穿过密度梯度层的沉降或上浮来达到的。
梯度液的密度随着离心本经的增大而增加。
密度梯度可以予形成，也可以在离心过程中自形成，经常，密度梯度的可以分为：速率一区带（Rate-30nal）离和等密度（Isopycnic）离心。
病毒空斑实验原理
病毒空斑实验原理
病毒空斑实验是一种常用的病毒鉴定方法，它通过观察植物叶片上的病毒感染
区域与未感染区域的差异，来确定植物是否感染了某种特定的病毒。
这种方法简单易行，且对于一些病毒的鉴定具有较高的准确性，因此在植物病毒学研究中得到了广泛应用。
病毒空斑实验的原理主要基于病毒感染后对植物叶片的影响。
在实验中，首先
需要选择一种易感染的植物作为实验材料，然后通过接种方式将待检测的植物病毒接种到植物叶片上。
接种后，病毒会在植物叶片内部进行复制和扩散，导致叶片出现病斑或病斑区域的变化。
而在实验的对照组中，同样的植物叶片则不接种病毒，作为未感染的对照。
接下来，通过一系列的观察和分析，可以发现感染了病毒的植物叶片上会出现
病斑，而未感染的对照叶片则不会有这样的变化。
病斑的形态、颜色、大小等特征可以帮助鉴定出感染的病毒种类和数量。
通过比对实验组和对照组的差异，可以准确地确定植物是否感染了目标病毒。
除了观察病斑的形态特征外，还可以利用分子生物学方法对病毒进行进一步的
鉴定。
例如，通过提取病毒RNA或DNA，利用PCR扩增等技术进行特异性检测，可以进一步确认病毒的种类和数量。
这种综合应用观察和分子生物学方法的方式，可以提高病毒鉴定的准确性和灵敏度。
总的来说，病毒空斑实验是一种简单、有效的病毒鉴定方法，通过观察病斑的
形态特征和分子生物学方法的综合应用，可以准确地确定植物是否感染了目标病毒。
在植物病毒学研究中，这种方法对于病毒的鉴定和研究具有重要的意义，也为植物病害的防控提供了重要的技术支持。
病毒纯化和Dio标记实验步骤说明书
Supplementary informationAccess to a main alphaherpesvirus receptor, located basolaterally in the respiratory epithelium, is masked by intercellular junctionsJolien Van Cleemput, Katrien C.K. Poelaert, Kathlyn Laval, Roger Maes, Gisela S. Hussey, Wim Van den Broeck, Hans J. Nauwynck.Supplementary experimental proceduresEHV1 purification and Dio-labellingCulture fluids of EHV1-infected RK-13 cells were clarified by centrifugation at 60,000g for 2h at 4°C. The virus pellet was pooled onto a discontinuous OptiPrep™ gradient (Sigma-Aldrich, St. Louis, MO, USA) containing 10-30% (w/v) of iodixanol and centrifuged at 100,000g for 2.5h at 4°C. After centrifugation, purified opalescent virus bands were harvested at the interface of the 15% and 20% layers. To ensure efficient virus lipophilic labelling, the buffer was exchanged to HNE buffer (5 mM HEPES, 150 mM NaCl, 0.1 mM EDTA, pH 7.4) by the use of a 50K filter device (Millipore corporation, Bedford, MA, USA). While vortexing, 2 nM of 3,3’-Dioctadecyloxacarbocyanine perchlorate (Dio) dissolved in DMSO (Molecular probes, Oregon, USA) was added to the virus. Subsequently, unbound Dio was removed by centrifugation onto a MicroSpin™ G-50 fine column (GE Healthcare, Buckinghamshire, UK). The degree of Dio-labelled virus purity (>90%) was evaluated by simultaneous immunofluorescent staining of EHV1 gB with mouse monoclonal antibody 3F6 (kindly provided by Prof. U. Balasuriya, University of Kentucky, USA) and quantitative analysis by confocal microscopy.Tissue collection and processingRespiratory mucosal explant isolation and cultivationThe respiratory mucosa was stripped from the underlying cartilage and washed in PBS to remove excess blood. Tissues were cut into small square pieces (25 mm2), placed with the epithelial side facing upwards onto fine-meshed gauzes and cultured in a 37°C, 5% CO2, humidified incubator for 24h at air-liquid interface in serum-free medium containing DMEM/RPMI (Invitrogen, Paisley, UK), supplemented with 0.1 mg/mL gentamicin, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B.EREC isolation and cultivationTracheae were trimmed upon arrival in the lab and washed in PBS to remove excess blood. Tissues were submerged into an enzyme mix of 1.4% pronase (Roche Diagnostics Corporation, Basel, Switzerland) and 0.1% deoxyribonuclease I (Sigma-Aldrich) in calcium- and magnesium-free PBS supplemented with 0.45% glucose (VWR International, Leuven, Belgium), 1% sodium pyruvate (Invitrogen), 100 U/mL penicillin and 0.1 mg/mL streptomycin for 48h at 4°C. Detached cells were then incubated in DMEM/F12 (Invitrogen), containing 1% MEM non-essential amino-acids (Invitrogen), 2.4 µg/mL insulin (Sigma-Aldrich), 100 U/mL penicillin and 0.1 mg/mL streptomycin in a plastic petri dish for 2h to reduce fibroblast contamination by adherence. Isolated EREC were either seeded immediately or stored in liquid nitrogen at a density of 2∙106cells per cryovial until further use. EREC were seeded at a concentration of 1.8∙106 cells/insert overnight into type IV collagen-coated (Sigma-Aldrich) 0.4µm pore size transwell cell culture wells (Costar, Corning, Fisher Scientific, Fair Lawn, USA) in DMEM/F12 (Invitrogen), supplemented with 5% non-heat inactivated FCS (Invitrogen), 1% MEM non-essential amino-acids, 100 U/mL penicillin, 1 mg/mL streptomycin, and 1.25 µg/mL amphotericin B. The next day, seeding medium was removed and the bottom platewells were filled with DMEM/F12, containing 2% Ultroser G (Pall Life Sciences; Pall Corp., Cergy, France), 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 1.25 µg/mL amphotericin B (EREC medium). The transwell, comprising the apical surface of the EREC, was left empty to mimic an air-liquid interface. EREC were incubated in a 37°C, 5% CO2 humidified incubator and medium was changed every 1-2 days until full differentiation. After 5-7 days, the EREC attained a trans-epithelial electrical resistance (TEER) of ~500-700 Ω∙cm-2. TEER was measured using an epithelial voltohmmeter (Millipore). The net resistance was calculated by subtracting the background resistance and multiplying the resistance by the surface area of the membrane.Disruption of intercellular bridges of respiratory mucosal explantsFirst, 24-well culture dishes were filled with 1 mL of a solution containing 50% sterile 3% agarose (low temperature gelling; Sigma-Aldrich) and 50% 2X MEM (Invitrogen). Explants were placed onto the solidified agarose with the epithelial surface facing upwards. Additional agarose was added until the lateral surfaces of the mucosa were fully occluded. Explants were then exposed for 1h at 37°C to different drugs (8 mM EGTA, 500 mM NAC, 20 mM DTT or 50 mM β-mercaptoethanol in PBS). PBS supplemented with calcium and magnesium was used as a control. Finally, explants were washed 3 times to remove excess drugs and were fixed in phosphate-buffered 3.5% formaldehyde solution, either immediately or after an additional 24h incubation. An automated system was used for paraffin embedding of the samples (Thermo Scientific™ STP 120 Spin Tissue Processor). Eight µm paraffin sections were first deparaffinised in xylene, then rehydrated in descending grades of alcohol, subsequently stained with haematoxylin-eosin, dehydrated in ascending grades of alcohol and xylene and finally mounted with DPX (Sigma-Aldrich). Ten pictures on five different sections per treated explant were taken with an Olympus IX50 light microscope fitted with 40X objective. The percentage of intercellular space in the epithelium was measured using ImageJ software (ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA). The region of interest (ROI, i.e. the epithelium) was drawn manually for each picture in the “ROI manager tool”. Next, the threshold value to distinguish blank spaces from cellular material was determined and the percentage of blank spaces between the cells (i.e. the intercellular space) was calculated. Immunofluorescent staining and confocal microscopyRespiratory mucosal explantsSixteen µm thick cryosections were cut using a cryostat at -20°C and loaded onto 3-aminopropyltriethoxysilane-coated (Sigma-Aldrich) glass slides. Slides were then fixed in 4% paraformaldehyde for 15min and subsequently permeabilized in 0.1% Triton-X 100 diluted inPBS. Non-specific binding sites were blocked by 15min incubation with avidin and biotin (Invitrogen) at 37°C. To label late viral glycoproteins, a polyclonal biotinylated horse anti-EHV1 was used for 1h at 37°C1, followed by incubation with streptavidin-FITC® (Invitrogen) for 1h at 37°C. The basement membrane of the tissues was stained with monoclonal mouse anti-collagen VII antibodies (Sigma-Aldrich), followed by secondary Texas Red® labelled goat anti-mouse antibodies (Invitrogen). Nuclei were detected by staining with Hoechst 33342 (Invitrogen). Slides were mounted with glycerol-DABCO and analysed using a Leica (TCS SPE) confocal microscope. The total number of plaques was counted on 50 cryosections and plaque latitude was measured using the Leica confocal software package. Five cryosections per explant were completely photographed and the percentage of infection in the epithelium (i.e. ROI) was determined using Image J software. The ROI (i.e. the epithelium) was drawn manually for each picture in the “ROI manager tool”. Next, the threshold value to distinguish the FITC positive signal from the background signal was determined and the percentage of FITC positive signal (i.e. infection) was calculated.Cellular glycosaminoglycans, heparan sulfate, chondroitin sulfate A and B and sialic acids were respectively stained with a monoclonal mouse anti-heparan sulfate antibody (10E4; Ambsio, Abingdon, UK), a monoclonal mouse anti chondroitin sulfate (CS-56; Bio-rad; Oxford; UK) or biotinylated Maackia Amurensis lectin (Vector Laboratories; Peterborough; UK) followed by a goat anti-mouse FITC® antibody or streptavidin-FITC®.ERECAntibodies were incubated directly in the transwells for 1h at 37°C. Cells were first incubated with a 1:1,000 dilution of a polyclonal rabbit anti-IEP antibody, kindly provided by Dr. D. O’Callaghan, Louisiana State University, USA. The diluent used was PBS containing 10% negative goat serum. This was followed by incubation with a goat anti-rabbit IgG FITC®conjugated antibody (Invitrogen). Nuclei were counterstained with Hoechst 33342 for 10minat 37°C. Transwell membranes were excised from the culture inserts and mounted on glass slides using glycerol-DABCO. Slides were examined using a Leica confocal microscope. The total number of plaques was counted on 5 random fields of approximately 3∙104 cells per insert. Plaque latitude was measured on 10 individual plaques using the Leica confocal software package.Enzymatic removal of cell surface N-linked glycans and sialic acids prior to EHV1 inoculationPNGase F (New England Biolabs, Ipswich, UK) removes complex, hybrid and oligomannose N-glycosylations and was applied onto apical or basolateral EREC surfaces for 12h at a concentration of 25,000 U/mL, diluted in EREC medium, supplemented with 10% glycobuffer (New England Biolabs; Ipswich; UK). Neuraminidase from Vibrio cholera (Sigma-Aldrich) has a broad substrate spectrum for sialic acids and was used for 1h at 50 mU/mL in PBS. The influenza A/Equine/Kentucky/98 (H3N8) strain served as a positive control during neuraminidase treatment of EREC. The virus was propagated in embryonated eggs and titrated onto Madin-Darby canine kidney (MDCK) cells. Influenza A strains can easily infect MDCK cells through interaction with cell-associated sialic acids, which can be removed with neuraminidase2. Equine influenza virus (EIV) A nucleoprotein was stained with the mouse monoclonal antibody HB-65 (ATCC) and subsequently visualized with FITC®-labelled secondary goat anti-mouse IgG antibodies. Nuclei were counterstained with Hoechst 33342 and coverslips were mounted using glycerol-DABCO. The percentage of positive cells was calculated based on the total number of positive cells out of 300 randomly selected cells. Correct cleavage of the respective sialic acids was corroborated by immunofluorescent staining of EREC with biotinylated Maackia Amurensis Lectin II (Vector laboratories). The complex was subsequently stained with Streptavidin-FITC® (Invitrogen). Ten z-stack confocal pictures were taken to distinguish apical from basolateral treatment. The means of the fluorescent apicalor basolateral signals were compared with Image J software and dropped significantly after treatment with neuraminidase, compared to control. Following enzymatic treatment, cells were washed 3 times with DMEM/F12 and the inoculum was delivered on top of the respective surfaces for 1h at 37°C. Concurrently, and as a positive control for enzymatic treatment, MDCK cells were treated with neuraminidase before inoculation with EIV. After 1h inoculation, unbound virus particles were removed by washing and cells were incubated for 10 hours before fixation in methanol, as described above.Supplementary figuresFigure S1. Cell viability in respiratory mucosal explantsTUNEL-staining data of tracheal ME (left) and nasal ME (right) after different treatments. Three independent experiments were performed and data are represented as means + SD. The lower case letters indicate significant (P<0.05) differences in the epithelium, while the upper case letters indicate significant differences in the lamina propria.Figure S2. The disruption of intercellular bridges in EREC(a) Trans-epithelial electrical resistance of EREC prior to treatment and after 30min treatment with PBS (control) or EGTA. Three independent experiments were performed and the data are represented as means + SD. Different letters indicate significant (P<0.05) differences. (b) EMA-staining confirmed no significant (P<0.05) differences in cell viability after different treatments, when compared to cell viability prior to treatment. Three independent experiments were performed and data are represented as means + SD. Different letters indicate significant (P<0.05) differences.Figure S3. Validation neuraminidase assay(a) During enzymatic treatment, MDCK cells were similarly pre-treated with neuraminidase before inoculation with EIV. Cells were fixed at 6hpi in methanol and EIV-positive cells were visualized using the monoclonal HB65 antibody. Five random fields of approximately 100 cells were screened to calculate the percentage of EIV-positive cells. Experiments were performed in triplicate. Data are represented as means + SD and different letters represent significant differences. (b) Correct sialic acid-cleavage from EREC surfaces was confirmed by confocal analysis of 10 different z-stacks per treatment. EREC were grown to confluency on transwells and treated at either the apical surface (left) or basolateral surface (right) with neuraminidase or control PBS for 1h at 37°C. Cells were then fixed in PFA and permeabilized in Triton X, sialic acids were stained with biotinylated Maackia Amurensis lectin and the percentage of fluorescent signal in either the apical or the basolateral domain of EREC was calculated. Data are represented as mean + SD. Significant differences after apical exposure are indicated by different lower case letters and after basolateral exposure by different upper case letters.Supplementary references1 van der Meulen, K., Vercauteren, G., Nauwynck, H. & Pensaert, M. A local epidemicof equine herpesvirus 1-induced neurological disorders in Belgium. Vlaams Diergeneeskundig Tijdschrift72, 366-372 (2003).2 Stray, S. J., Cummings, R. D. & Air, G. M. Influenza virus infection of desialylatedcells. Glycobiology10, 649-658 (2000).。
空斑实验简介
3、空斑纯化3.1病毒TCID501）在离心管中用MEM将病毒液作连续10倍的稀释，从10-1-10-6。
2）将稀释好的病毒接种到96孔培养板中，每一稀释度接种一纵排，共8 孔，每孔接种100µl。
3）在每孔加入细胞悬液100µl，使细胞量达到2~3×105个/ml。
4）设正常细胞对照，正常细胞对照作两纵排。
（100µl生长液+100µl细胞悬液）5）逐日观察并记录结果，一般需要观察5-7天。
6）结果的计算，按Reed-Muench法。
Reed-Muench法距离比例＝（高于50%病变率的百分数-50%）/（高于50%病变率的百分数-低于50%病变率的百分数）＝（91.6-50）/（91.6-40）＝0.8lg TCID50=距离比例×稀释度对数之间的差+高于50%病变率的稀释度的对数=0.8×(-1)+(-3)=-3.8TCID50=10-3.8/0.1ml含义：将该病毒稀释103.8接种100µl可使50%的细胞发生病变。
3.2中性红溶液和DMEM营养琼脂的配制（1）中性红溶液的配制1）取中性红1g、NaCl 8.5g溶于1L超纯水中2）高压灭菌后分装保存于4℃冰箱中。
（2）DMEM营养琼脂的配制1）取2g琼脂糖于100ml超纯水中，微波炉中火2min溶解后摇匀，高压灭菌。
2）取小牛血清4ml、中性红3.3ml无菌操作加到100ml双倍DMEM中，37℃水浴30min，摇晃均匀。
3）溶解的2%琼脂置于44℃水浴30min，与双倍DMEM混合液混合后，置于44℃待用。
3.3铺6六孔板(1) 将已准备好的V ero细胞消化均匀铺于6孔板中。
(2)细胞长成单层后吸去培养液，每孔加2ml无血清维持液置于37℃1h。
(3)将病毒用无血清维持液做10倍递增稀释。
(4)吸去孔内维持液，按合适稀释度每孔滴加200μl稀释好的病毒液, 每个稀释度加两个孔，置37℃吸附1小时后弃病毒液。
实验技术-空斑纯化方法步骤以及若干问题
材料和试剂：DMEM培养基，细胞板，中性红，琼脂糖，PBS或无抗无血培养基，1ml和10ml 移液管等。
操作步骤：1 PK15细胞铺细胞板，细胞密度要较高，才轻易形成空斑（但太高了也不好，有可能会形成很多层细胞，造成有细胞形成病变地方有其它层细胞没有坏死也被染色从而无法形成空斑）。
2 过夜后，用无抗无血培养基清洗洗吧，然后再进行病毒吸附，原液进行10倍倍比稀释，自己能够探索一下感染稀释度，通常病毒价较低时候，能够降低稀释度。
看空斑形成情况，要是空斑形成太大，连成片，就得增加稀释度。
3 吸附1-2小时，用无抗无血培养基（或无菌PBS）清洗2-3遍后配制第一次覆盖液。
使用1.6%琼脂糖（加热至液体状）和两倍DMEM溶液等体积混合，逐滴加到细胞板中。
没孔加入1-2ml覆盖液。
放置于37度培养箱中培养。
4 天天观察细胞病变情况，出现显著病变时进行二次覆盖，覆盖液和第一次差不多，只是要加入细胞染色液中性红（0.1%），通常体积比为10ml：10ml：1ml （能够合适增加一点，假如染色效果不良话）。
5 染色5H以上应该就能够观察染色情况，理论上伪狂犬病毒应能形成白色空斑而PCV2不会产生空斑。
在不知道PCV2在哪里情况下，我们随机挑取若干个没有空斑细胞，以后进行传代。
应该能够得到无伪狂犬污染PCV2病毒种。
1 蚀斑试验需要细胞长至什么程度能够接毒？显微镜下观察，通常情况下，铺满细胞板底部即可，这么不会造成空洞影响蚀斑观察。
不一样细胞不一样，通常时候是长满T25细胞传一个六孔板，第二天就接毒。
2 细胞上用什么覆盖物？琼脂？琼脂糖？低熔点琼脂糖？浓度应该是多大？我用是lonza企业低熔点琼脂糖，终浓度是0.75%。
感觉不错，就是挺贵，2k+人民币。
3 假如做挑斑纯化，是不是就不能够染色，染色后对毒力有何影响？不染色就能够看出来，因为培养基础身里面就含有酚红展现颜色，形成斑没有颜色，能够直接挑斑；我也染色后挑过，感觉影响不大，可能不一样病毒不一样吧。
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EV71空斑纯化实验protocal
原理
病毒感染细胞后，由于固体介质的限制，释放的病毒只能由最初感染的细胞向周边扩展。
经过几个增殖周期，便形成一个局限性病变细胞区，此即病毒蚀斑。
一个蚀斑是由最初感染细胞的一个病毒颗粒形成的，接种的病毒液要充分分散和稀释。
流程
1.铺板
将已长满80~90%RDcell消化均匀铺于6孔板中。
2.病毒感染
细胞长成单层达80~90%吸去培养液，将病毒做10倍稀释，共做5个梯度，并向每孔中加入200μL稀释好的病毒液（阴性对照加DMEM200μL），置于37℃温箱吸附两小时后弃残液。
3.制备2%琼脂糖
使用低熔点琼脂糖配方：0.2g加入10ml 无菌PBS中，121℃高压灭菌15min，取出后置于55℃水浴锅中待用
4.混合
将上述琼脂糖和2X？维持液(FBS浓度为2%）40~50℃水浴1:1混合后，加入培养板各孔，每孔2ml,室温放置30min以上使其冷却凝固成覆盖层
5.培养
把培养板倒置？，在37℃二氧化碳培养箱继续培养48h。
6.染色
制备含0.2%结晶紫(生理盐水稀释）的2%琼脂糖：双倍维持液（1：1），置于40~50℃水浴待用，吸取上述混合液加入培养板各孔，每孔2ml,使冷却凝固成第二覆盖层
7.培养
把培养板倒置，在37℃二氧化碳培养箱继续培养48h观察，每皿蚀斑数量＜10个的稀释度中，挑选附近10mm均为健康的蚀斑。
8.将挑选的病毒至少再传2代
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