Name: isolation of viral replication compartment-enriched sub-nuclear fractions from adenovirus-infected normal human cells
Uploaded: 2015-11-12T14:00:00.000+00:00
Duration: 10 min 22 s
Description: Watch this Scientific Journal Video about Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells at JoVE.com

This work was supported by grants from CONACyT-SEP (SEP-2008-84582; CB-2011-01-168497) and Promep-SEP for R.A.G.; P.H. received a scholarship from CONACyT (447442).

1. HFF细胞培养和Ad-感染<ol><li>传播的Ad5 WT病毒在HEK-293细胞和滴度荧光形成单元（FFU）上HFF细胞单层如前面8中所述 。 </li><li>生长人类包皮成纤维细胞（HFF）的10ml DMEM / 10％胎牛血清（FBS）中无菌培养100mm平皿在37ºC和5％ 的 CO 2的加湿培养箱中。确定使用Neubauer室通过在4个16平方集计数细胞的细胞数量。通过计算细胞在4个16平方集的平均数和10 4乘以该数而得到每毫升细胞数，对于包括在步骤1.3每次感染后，用1×10 7 HFF细胞。 </li><li>模拟侵染或感染HFF细胞在用1ml的Ad5的在每个培养皿的DMEM，在30聚焦的MOI 100mm组织培养皿5型腺病毒（Ad5的）野生型（WT）（H5pg4100 8）形成单位，或FFU每个细胞。孵育2小时啊umidified细胞培养培养箱中37ºC和5％的CO 2，小心摇动菜每15分钟，以确保病毒接种物在细胞上均匀分布。在此时间后，除去培养基并加入新鲜的DMEM补充有10％FBS和在湿润的细胞培养培养箱孵育16，24或36小时，在37℃，并 5％的CO 2。继续执行步骤2.1。 </li></ol> 2.制备亚核馏分富含有腺病毒RC <ol><li>收获的Ad5感染或模拟感染HFF细胞与细胞刮收集细胞在无菌离心管。确定细胞数如在步骤1.2。使用1×10 7个细胞用于在步骤1.3中指定的每个时间感染后。 </li><li>离心细胞，在220×g离心，4ºC5分钟。 </li><li>重悬在冰冷的PBS将细胞沉淀（137毫摩尔NaCl，2.7毫米氯化钾，10mM的磷酸氢二钠和1.8mM的KH 2 PO 4）。洗涤细胞沉淀S使用5毫升冰冷PBS每次洗涤3次。为了这个目的，离心细胞，在220×g离心，4ºC5分钟，倾析并弃去上清液（SN），并且重悬细胞沉淀通过温和移液。 </li><li>破坏细胞膜，重悬在700细胞沉淀μ升冰冷的低渗缓冲液（10mM HEPES pH为7.9，10mM的氯化钾，1.5mM的MgCl 2的，0.5mM的DTT，20μ微克/毫升苯甲磺酰氟（PMSF） ，半胱氨酸，丝氨酸，苏氨酸和天冬氨酰蛋白酶抑制剂的混合物包括10μ克/毫升牛胰蛋白酶抑制剂，10μ微克/毫升胃蛋白酶抑制素A和10μ克/ 毫升 N-乙酰基-L-亮氨酰-L-亮氨酰-L- -argininal），并让细胞膨胀在冰上3小时。 </li><li>裂解使用均化器以宽松的A型聚四氟乙烯杵的细胞。执行80 DOUNCE笔画和监视器样品每20笔通过亮视野显微镜，以确保所有细胞已被溶解，但该核具有n个加时赛被损坏或破裂。 </li><li>离心细胞匀浆在300×g离心，4ºC5分钟。存储SN作为在-20 C在离心管中的胞质级分。 </li><li>从细胞核除去细胞碎片，重悬沉淀在750μ升溶液1（S1）的（0.25M蔗糖，10mM的MgCl 2的20μ克/毫升PMSF，半胱氨酸，丝氨酸，的混合物苏氨酸和天冬氨酰蛋白酶抑制剂，包括10 μ克/毫升牛胰蛋白酶抑制剂，10μ微克/毫升胃蛋白酶抑制素A和10μ克/ 毫升 N-乙酰基-L-亮氨酰-L-亮氨酰-L- argininal）和层以上的溶液2等体积（S2） （0.35M的蔗糖，0.5mM的MgCl 2的，20μ克/毫升PMSF，半胱氨酸，丝氨酸，苏氨酸和天冬氨酰蛋白酶抑制剂，包括10μ克/ ml牛胰胰蛋白酶抑制剂，10μ微克/毫升胃蛋白酶抑制素A和10μ的混合物克/ 毫升 N-乙酰基-L-亮氨酰-L-亮氨酰-L- argininal）。 CENTRifuge在1400×g离心，4ºC5分钟。 </li><li>丢弃使用微量的SN。避免干扰颗粒。 </li><li>重悬在含有750μ升S2的分离的细胞核沉淀。 </li><li>为了分离富含腺病毒RC（RCF）亚核分数，超声处理悬浮细胞核用超声波浴，用两个5分钟的脉冲，或直到所有核裂解通过亮视野显微镜观察到。根据需要将样本保持在或低于4℃在超声浴用冰。 </li><li>层的超声处理的核以上的溶液3（S3）（0.88蔗糖，0.5mM的MgCl 2的）和离心机以3,000×g离心，4ºC10分钟的等体积。存放1.5毫升上清液作为核质馏分（NPL）在-70ºC。悬浮颗粒在700μ升S2的和存储的RCF在-70ºC。 </li></ol> RCF 3. Western印迹分析注:对于不良贷款和RCF分数西部印迹分析等预留640 微升的不良贷款和300μl对于RCF从步骤2.11中获得的总体积。 <ol><li>混合15μ升在Laemmli缓冲液2倍每个亚核级分（4％SDS，20％甘油，10％2-巯基乙醇，0.004％溴酚蓝，0.125摩尔Tris盐酸pH6.8）中并煮沸，在95°C下5分钟。加载样品中10％的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）变性凝胶和分离蛋白1.5小时，在20毫安（mA）的。 </li><li>通过以400mA电印迹1.5小时到聚偏二氟乙烯（PVDF）膜转移蛋白。 </li><li>方框膜进行2小时，在使用3％脱脂奶粉的PBS室温。 </li><li>孵育O / N在4ºC与针对病毒的E2A DNA结合蛋白（B6-8 9）第一抗体以1:500稀释在PBS / 0.3％脱脂奶粉。 </li><li>洗涤膜3次，用PBS / 0.1％吐温20 10分钟。 </li><li>孵育小鼠抗IgG二的膜耦合到辣根过氧化物酶（HRP）tibody以1:10,000稀释在PBS中2小时，在室温。 </li><li>用PBS洗膜3次/ 0.1％吐温20 10分钟。 </li><li>开发使用增强的化学发光和X光片的膜。 </li></ol> 4.病毒DNA检测RCF 注:对于从不良贷款和RCF部分DNA的提取，使用210μl对于不良贷款和100μl对于在步骤2.11中获得的总体积的RCF。 <ol><li>在55ºC孵育子核级分1小时用蛋白酶K为1mg / ml和0.5％吐温20。 </li><li>通过温育样品10分钟，在95℃下失活的蛋白酶K。 </li><li>离心样品在20000×g离心，在室温2分钟。 </li><li>收集SN。沉淀DNA与1/10体积的3M醋酸钠和异丙醇中的一个体积，O / N在4℃下。 </li><li>离心样品在20000×g离心，在室温10分钟。 </li><li>丢弃SNü唱微量。在20000×g离心，4℃5分钟洗涤用70％乙醇，离心沉淀。 </li><li>重悬的DNA在10μ升的10mM的Tris-盐酸pH 7.4的</li><li>使用分光光度计通过测量光密度（OD）在260nm处定量的DNA。 </li><li>为了扩增病毒DNA，使用100ng的DNA的使用Taq DNA聚合酶在标准PCR反应各亚核级分在25μ升反应体积的用引物，其允许主要晚期转录单位内的区域的放大，从核苷酸7273至核苷酸7353（81基点）（FW:GAGCGAGGTGTGGGTGAGC; RV:GGATGCGACGACACTGACTTCA）。使用下面的循环条件:初始变性3分钟，在95°C下，之后20个循环的扩增（在95°C下，退火1分钟，在62ºC和延伸30秒，在72ºC1分钟）和用于最终延伸步骤3分钟72ºC。 </li><li>运行PCR产物在2％琼脂糖凝胶上，在90V。色漆乙锭溴化物在0.5μ克/毫升的终浓度和可视化的DNA以证实病毒DNA富集区域合作框架。处理溴化乙锭格外小心，并始终确保要戴手套，因为这种化合物是一种有效的诱变剂。根据制度规定进行处置的溴化乙锭。 </li></ol> 5.晚期病毒mRNA检测的RCF 注:对于从不良贷款和RCF部分RNA分离，用640μl对于不良贷款和300μl对于RCF从步骤2.11中获得的总体积。 <ol><li>使用Trizol根据制造商的说明书分离自亚核级分的RNA。重悬RNA在50μ升DEPC（diethilpyrocarbonate）处理过的水。 </li><li>量化使用分光光度计测量的OD 260nm处的核糖核酸（大约3μg的获得）。存放RNA在50毫微克/ 微升分装置于-70ºC。 </li><li>检查纯化RNA为不存在DNA污染的通过进行在不存在逆转录酶（RT）的控制PCR反应中，用5纳克总RNA，并且在步骤4.9中描述的循环的条件。 <ol><li>如果DNA污染不存在任何扩增子应来制造。如果DNA污染存在，孵育样品与10单位的DNase I U RNA酶抑制剂，10，0.1M的Tris-HCl pH值8.3，0.5M KCl和15mM的MgCl 2的30分钟，在37ºC。继续执行重新分离RNA如在步骤5.1。 </li></ol></li><li>分析相关的RCF病毒mRNA，放大100纳克RNA的通过RT-PCR，使用设计用于检测从不同的基因家族（表1）的腺病毒晚期mRNA的引物各亚核级分。 <ol><li>进行反转录（RT）下，用M-MuLV的逆转录酶在20μ升反应容积为1小时，在42ºC并在70ºC10分钟用100纳克的RNA，来自每个亚核级分在一个标准的RT反应。 </li><li>对于扩增，使用1μ升在PCR反应中，将cDNA的如在步骤4.9。使用下面的循环条件:初始变性3分钟，在95°C下，接着扩增25个循环（在95°C下1分钟，退火在30秒，在72ºC 于表1和延伸规定的温度1分钟）和一为3分钟，在72ºC最终延伸步骤。 </li></ol></li><li>运行RT-PCR产物在2％琼脂糖凝胶上，在90五染色凝胶用溴化乙锭，在0.5μ微克/毫升的最终浓度和可视化，以证实病毒晚期mRNA关联到RCF。处理溴化乙锭如在步骤4.10所示。 </li></ol> RCF 6.免疫可视化注:进位出这个过程在层流柜，以避免样品污染的任何尘埃颗粒，并在使用前过滤所有的解决方案。 <ol><li>现货5 微升步骤2.10可怕获得的RCF分数ctly上的硅烷涂覆的滑动。 </li><li>让光点干约5分钟，在室温。 </li><li>重新水合物缓慢和间接吸取PBS 500μL的RCF点旁边的降低，且让它通过倾斜滑动流动。从侧面流掉过量PBS中。 </li><li>由覆盖用500μ升的PBS / 5％牛血清白蛋白（BSA）的样品进行2小时，在室温块。 </li><li>轻轻和间接吹打的PBS 500μ升到样品洗载玻片3次。倾斜滑动流掉过量PBS中。 </li><li>孵育针对病毒的E2A DNA结合蛋白（B6-8 9）在一个1第一抗体样品1:500稀释在PBS中2小时，在室温。覆盖20μ升抗体溶液的点样的样品，孵育在潮湿室中，以避免干燥。 </li><li>用PBS / 0.02％吐温20轻轻和间接吹打500μ升的PBS到样品洗样品3次。倾斜滑动流掉前塞斯洗涤液。 </li><li>孵育样品与耦合到该被激发在488nm以1荧光团的小鼠抗IgG第二抗体:在4ºC2000稀释在PBS中，1小时。覆盖20μ升抗体溶液的点样的样品，孵育在潮湿室中，以避免干燥。 </li><li>用PBS / 0.02％吐温20轻轻和间接吹打500μ升的PBS到样品洗样品3次。倾斜滑动以排干多余的洗涤溶液中。 </li><li>还包括与使用2μ升的PBS / 10％甘油作为安装平台盖玻片载玻片上的点样的样品。轴封采用透明指甲油和存储人-20ºC。 </li><li>分析使用荧光显微镜的样品中，用63×物镜和一个1.4数值孔径（NA），在488nm处的波长。 </li></ol>

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The authors have nothing to disclose.

In order to elucidate the molecular mechanisms that govern regulation of cellular activities by viral infection understanding the composition and activities associated with RC would be instrumental. Therefore, to make a detailed analysis of RC, we established a cell-free system that takes advantage of the size and biochemical composition of these virus-induced structures, to isolate subnuclear fractions enriched with RC using a simple procedure that relies on velocity gradients with sucrose cushions. Critical steps of the procedure that require standardization depending on the cell type used are: i) standardization of the times used for cell swelling to avoid disruption of nuclei; ii) formation of the sucrose gradients so that fractions can be properly separated; iii) constant monitoring of the samples throughout the procedure by bright field microscopy; iv) sonication time and intensity to ensure that all nuclei are lysed but RC are not disrupted; v) all fractionation steps should be carried out on ice to avoid disruption of nuclear structures. 
Limitations of the technique that can be encountered or can be anticipated are: i) that nucleoli and perhaps other subnuclear structures or domains that share similar dimensions and biochemical composition are coisolated in the RC fraction, making the relative abundance of replication compartments versus other similar nuclear bodies variable depending on the virus, cell type and time post-infection used for their isolation; ii) it is possible that just as the size and composition of RC changes as the replication cycle of the virus progresses, the compactness of the RC particles may also change, making it necessary to perform experiments designed to determine detailed ultrastructural changes of the RC particles at different times post-infection to evaluate not only changes in the particles composition and activities, but whether their stability may vary. While these issues may limit the characterization of RC composition and activities at various different times post-infection, as less stable RC obtained by this procedure may not recapitulate their counterpart in the infected cell nucleus, such limitations may be overcome by using higher multiplicities of infection or by determining the times post-infection at which viral RC have attained a minimum size and abundance in the infected cell nucleus. These potential limitations notwithstanding, the technique reported here provides several advantages over the analysis that has hitherto solely relied on microscopy; namely that the precise biochemical and functional analysis of RC can be performed using this novel approach. 
We show here that the RC-fractions (RCf) obtained are enriched with bona fide RC markers, such as the viral E2-72K DBP protein, viral DNA and RNA. Using biochemical analysis by immunoblotting and PCR, we have confirmed that this approach facilitates the detailed analysis of components and activities associated to RC and should help unravel molecular mechanisms that regulate virus-host cell interactions in the infected-cell nucleus. 
It is well established that DNA replication, transcription and post-transcriptional processing of viral late mRNA are closely associated to viral replication compartments, events that lead to viral progeny production2,11,12. In adenovirus-infected cells the onset of viral DNA replication marks the transition to the late phase of infection and results in the activation of the Major Late promoter, which directs the production of high quantities of viral late transcripts. However, the L4 genes are expressed through a novel promoter (L4P) that is independent from the MLP13,14. Besides this complex program of late promoter activation, the selection of 3&#8217; splice sites for longer introns is progressively favored as the late phase proceeds. In addition, production of these transcripts is regulated by their selective release from the site of synthesis and by selective export to the cytoplasm. 
However, regulation of the late promoters and molecular events that lead to the selective production of viral late mRNA is incompletely understood. Therefore, as a proof of concept for the procedure reported here, we demonstrate that viral DNA and viral late mRNA can be isolated in RCf, suggesting that these subnuclear fractions can be used to determine the pattern of synthesis and partitioning of different species of viral late mRNA, hence allowing the use of quantitative techniques and potentially advancing the detailed study of the complex pattern of viral late mRNA biogenesis. 
Furthermore, this technique can be used to study the molecular mechanisms that are responsible for the efficient replication of the viral genome, regulation of the complex program of viral gene transcription and cellular factors that are co-opted in RC, as well as morphological and ultrastructural studies of these viral induced structures. Moreover this system should be easily adapted to isolate RC from other nuclear replicating viruses, and should help unravel viral mechanisms that govern virus-cell interactions.

腺病毒包含复制在被感染细胞核的双链DNA基因组。当病毒DNA进入细胞核，它局部化相邻的PML核机构1。随着病毒的早期基因的表达，核架构大幅改组，诱导形成病毒性微环境，被称为病毒复制车厢（RC）2。由于腺病毒（广告）RC是网站，病毒基因组复制和病毒晚期基因表达发生，他们提供了一个环境，招募所有参与这些过程中必要的病毒和细胞的因素。有趣的是，各种细胞蛋白负责细胞抗病毒反应，如DNA损伤反应的，先天免疫应答和抑制肿瘤是增选到这些病毒网站2。因此，广告RC可以考虑监管中心之一，促进高效病毒复制，同时伴随调节蜂窝的抗病毒反应，表明这些结构是关键的病毒 - 宿主细胞相互作用的理解。尽管如此，钢筋混凝土形成的分子机制，其组合物和相关活动是知之甚少。 腺病毒的RC，以及RC从复制在核中的其他DNA病毒不相关联的膜，而相比之下，细胞质的RC 3。此外，这些病毒诱导的结构是可能的蛋白质和核酸的待全部由。 RC形成在感染的细胞的RNA病毒（通常被称为病毒工厂）已被分离，同时它们的细胞质定位的和膜结合的状态，这促进了对它们的详细的形态，功能和生化特征4的优势。 据我们所知，核病毒的RC没有被隔离，这可能是由于所述的核内米的核结构和不存在的复杂性embranes这将有利于他们的孤立。他们的研究一直依靠而不是在免疫荧光显微镜，荧光原位杂交和透射电子显微镜。然而，尽管固有的隔离亚核结构复杂，其它核领域，如核仁和Cajal间机构一直在5,6前隔离。因为核仁和RC都是由蛋白质和核酸的，并且具有0.5之间的直径 - 5微米，我们推测，RC也应该适合于隔离。因此，为了更精确地表征相关联的RC的分子组成和功能，我们建立了一种新方法，以分离富含RC亚核级分。为此，我们制备使用速度梯度和蔗糖垫类似程序用于分离核仁7或其他核结构域6子核级分，并建立了无细胞系统，使分子组合物的研究和有关的活动RC。因此这种技术应该提前的病毒 - 宿主细胞相互作用的理解，并表示一个强大的工具，也应有助于RC的详细分析从该复制在核中的其他病毒和诱导形成类似的尺寸的复制隔间的那些形成在腺病毒感染的细胞，如，疱疹病毒，乳头状瘤病毒或多瘤病毒。

<table><tbody><tr><td>DMEM</td><td>Gibco</td><td>12100-046</td><td>Warm in 37 &amp;ordm;C water bath before use</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>12484-028</td><td></td></tr><tr><td>Sucrose, Ultra Pure</td><td>Research Organics</td><td>0928S</td><td>Prepare a 2.55 M stock solution and store at 4 &amp;ordm;C</td></tr><tr><td>Dounce homogenizer</td><td>Kontess Glass Company</td><td>884900-0000</td><td></td></tr><tr><td>Branson 1800 Ultrasonic Bath</td><td>Branson</td><td>Z769533&amp;nbsp;SIGMA</td><td>Turn on 15 min before use.</td></tr><tr><td>Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Life Technologies</td><td>A-21121</td><td>Use at a 1:2,000 dilution in PBS</td></tr><tr><td>Silane-Prep Slides</td><td>Sigma</td><td>S4651-72EA</td><td>Open in a laminar flow cabinet</td></tr><tr><td>SuperSignal West Pico Chemiluminescent Substrate</td><td>Pierce ThermoScientific</td><td>34080</td><td></td></tr></tbody></table>

isolation of viral replication compartment-enriched sub-nuclear fractions from adenovirus-infected normal human cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

因为病毒复制室（RC）是蛋白质和核酸，类似于其他核结构域组成的亚核病毒诱导的结构，他们证明了通过基于生物化学特征速度梯度以便能够进行隔离。在分馏协议关键步骤示于图1.在每一步骤中，样品需要通过亮视野显微镜进行监测，以确保不同的亚细胞级分的完整性。例如，肿胀的细胞时，需要在低渗缓冲液孵育时间以溶胀细胞质避免了对细胞核进行标准化。细胞均化，完整的细胞核，自由细胞质成分包括内质网的膜后，需要获得。而且，声波处理时间的需要，以破裂所有细胞的核膜不中断的RC标准化。 获得子核级分之后，键控件需要包括确定区域合作框架的关联和善意 RC标志物富集。腺病毒的RC使用抗体针对病毒的E2A-72K蛋白（DBP）通常显现在感染细胞的免疫荧光。舒张压是一种病毒蛋白直接参与病毒基因组的复制;因此，舒张压在颗粒中的存在富集在RCF演示此病毒蛋白与孤立粒子的直接关联， 如图1，此外，检测舒张压在RCF通过Western印迹的证实这种蛋白质的分离的关联RC。 在图2中示出，通过后期倍感染后（36 HPI），DBP富集在RCF相比，核质馏分（NPL），这表明RC与此过程在不同的时间感染后反映预期的时间得到DBP协会RC模式。钢筋混凝土的主要病毒组分是病毒DNA ITSELF。在这样的实验中，如图3所示，我们表明，增加量与RCF病毒DNA准作为病毒复制循环的进行，这表明，对于DBP，DNA复制在这些部分的时间模式，也可以研究。 除了含有病毒蛋白和病毒基因组，RC也病毒晚期基因表达的位点。在图4中 ，我们提出的设计通过RT-PCR测量各种物种病毒晚期mRNA，并在36 HPI RCF和NPL之间的隔离的水平实验的代表性结果。病毒晚期mRNA合成在RC和他们postranscriptional处理在这些网站发起;之后，这些病毒mRNA从RC分离，被释放出来的核质，并随后出口到细胞质中。使用放大三方领导人的​​外显子交界的引物成熟的病毒晚期mRNA的总池（ML mRNA）的测量，一序列构成的5&#39;所有的腺病毒晚期mRNA（ 图4A）的。外显子结在L2的具体成熟转录mRNA的，L4和L5的家庭也测量。已经确定，由于病毒复制循环的后期阶段的进展，增加量的病毒晚期mRNA出口到细胞质;然而，由晚倍生产L5的mRNA种类的进一步与其他晚期mRNA家庭比较增加。在播种于图4中的代表性结果，在ML的mRNA的约2.5倍的差异是观察NPL与RCF相比，如预期。有趣的是，当我们从特定家庭比较的mRNA，L2和L4的mRNA似乎分布在这两个亚核级分（图4B 和C，分别地）之间相似水平，而与此相反，L5的mRNA显示在NPL几乎2倍的增加相比于RCF。这些结果表明，在SY差动模式<html> nthesis和解放的不同病毒晚期mRNA种腺病毒的RC（如已提出10之前）。显著，这些结果也表明的不同步骤中的病毒mRNA的生物合成的精确测量可以使用分离的RC与此新颖程序进行。 <img alt="图1" src="/files/ftp_upload/53296/53296fig1.jpg" /> 在分馏过程中各个步骤的图1明场显微术。在钢筋混凝土的隔离，样品需要通过光学显微镜进行监测，以确保亚细胞级分的完整性。该图显示了该过程中使用，以获得RCF的步骤，从HFF细胞的肿胀，对核的通过蔗糖垫分离和RCF的隔离。 40倍的显微照片:比例尺50μ米; 63X显微照片:比例尺5μ米; DBP显示为绿色。TPS://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53296/53296fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53296/53296fig2.jpg" /> 图2.西方反对DBP印迹的样品通过SDS-PAGE分析并处理对DBP，病毒性RC的真正标志物免疫印迹。以确定在RCF的蛋白质的富集，这是有用的存在和DBP的相对丰度在两个RCF和NPL在不同时间感染后比较。在HFF细胞16 HPI代表了腺病毒复制周期中的早期; 24 HPI标志着过渡到感染的病毒DNA合成开始的晚期; 36 HPI代表一晚的时间后感染。舒张压的预期分子量被示出。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53296/53296fig2large.jpg" target="_blank">请CLIC</a>ķ此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53296/53296fig3.jpg" /> 图3. PCR检测病毒DNA（VDNA）的RCF。DNA的RCF和不良贷款进行纯化，在24和​​36 HPI。病毒DNA，使用特异于病毒基因组的引物通过PCR扩增。该图显示病毒DNA在RCF在36 HPI的富集。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53296/53296fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53296/53296fig4.jpg" /> 。图4的病毒晚期的mRNA的RNA 分析从RCF和NPL分离并通过RT-PCR分析，以检测特定病毒晚期mRNA（A）总病毒晚期基因（ML:主要晚期）;（B）从L mRNA的2家;（C）的mRNA从L4家庭;（四）从L5家族基因。数值代表平均值±一式三份样品的标准偏差。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53296/53296fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" fo:keep-together.within-page="1"><tbody><tr><td>名称</td><td>正向引物序列（5&#39;-3&#39;） </td><td>反向引物序列（5&#39;-3&#39;） </td><td>退火温度</td></tr><tr><td> ML基因</td><td> GCCTCCGAACGGTACTCCGCC </td><td> CGCCACGGTGCTCAGCCTACC </td><td> 60ºC </td></tr><tr><td> L2的mRNA </td><td> GTCACAGTCGCAAGATGTCCAAGC </td><td> GCAACGCCAGCATGTCCTTATGC </td><td> 58ºC </td></tr><tr><td> L4基因</td><td> CCTCCGAACGGTACTCCGC </td><td> CCTTGCTCATCTTGCGACTGTG </td><td> 58ºC </td></tr><tr><td> L5的mRNA </td><td> GTCACAGTCGCAAGATGAAGCG </td><td> GGTAACTAGAGGTTCGGATAGGCG </td><td> 60ºC </td></tr></tbody></table> 表1.引物用于病毒晚期基因扩增的mRNA的ML:这些引物允许内的三联前导的区域的扩增，在5&#39;序列是共同的所有病毒晚期mRNA; L 2的mRNA的引物允许在PV的mRNA内的特定区域的放大; L4的mRNA的引物允许为100K的mRNA内的特定区域的放大; L5的mRNA允许区域内的纤维的mRNA的扩增。的顺序和退火温度为每个拘谨示器。

Watch this Scientific Journal Video about Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells at JoVE.com

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

isolation-viral-replication-compartment-enriched-sub-nuclear

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

8.7K 次观看.  Universidad Autónoma del Estado de Morelos. We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC. 

视频: Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

科研

免疫与感染

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

重组细小病毒与腺病毒派生系统的帮助下高效生产

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

A型流感病毒的核蛋白复合物的分离纯化及可视化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

生物学

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

腺病毒生产的有效方法

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

生物工程

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an ...

通过单步肝素亲和层析纯化 AAV 载体

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

作者聚焦：腺相关病毒载体生产和纯化的改进方法

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

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此视频中的章节

从核衣壳的病毒DNA的制备

重组细小病毒与腺病毒派生系统的帮助下高效生产

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

A型流感病毒的核蛋白复合物的分离纯化及可视化

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

成人的心肌细胞的分离和遗传操作共聚焦成像

腺病毒生产的有效方法

Construction of Adenoviral Vectors using DNA Assembly Technology

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体