BK-多瘤病毒非编码控制区域驱动转录活性通过流细胞测定的测量
1Department of Nephrology,University of Duisburg-Essen, University Hospital Essen, 2Institute for Virology,University of Duisburg-Essen, University Hospital Essen, 3Center for Translational Medicine, Medical Department I, Marien Hospital Herne,University Hospital of the Ruhr-University of Bochum, 4Department of Infectious Diseases, University of Duisburg-Essen,University Hospital Essen Hufelandstr
Summary
在本手稿中,提出了使用 HEK293T 细胞转染的基于 FACS 的 BK-多瘤病毒转录活性的测量方案,该细胞与表达 tdTomato 和 eGFP 的双向报告质粒转染。该方法进一步允许定量确定新化合物对病毒转录的影响。
Abstract
多瘤病毒,如BK-多马病毒(BKPyV),可引起免疫功能低下患者的严重疾病。然而,由于目前没有高效抗病毒药物,需要用测量潜在抗病毒药物影响的方法。在这里,一个双荧光报告器,允许分析BKPyV非编码控制区域(NCCR)驱动的早期和后期促进者活动,以量化潜在的抗病毒药物对病毒基因表达的影响,通过tdTomato和eGFP表达。此外,通过克隆BKPyV-NCCR增压,该议定书中从免疫功能低下的肾移植患者的血液衍生DNA中得到的,可以确定NCCR-重组对病毒基因表达的影响。在克隆患者衍生的氨基细胞后,HEK293T细胞与报告质粒转染,并用潜在的抗病毒剂进行治疗。随后,对细胞进行FACS分析,以测量转染后72小时的平均荧光强度。为了测试具有潜在细胞循环抑制作用的药物的分析,只使用转染的荧光细胞。由于这种测定是在大T抗原表达细胞中进行的,因此可以相互独立地分析早期和晚期表达的影响。
Introduction
聚瘤病毒代表一个独立的小双链DNA(dsDNA)病毒家族,以西米安病毒40(SV40)为原型物种。原发性感染主要发生在儿童期,通常无疾病症状,通常引起免疫宿主的潜在感染。BK-多瘤病毒(BKPyV)主要在肾小管细胞中持续存在,不会引起肾病,但是,在肾移植后免疫能力受损的情况下,病毒可以重新激活,并造成严重的损害和受损的移植物功能达到高病毒血症 (1 x 104 BKPyV DNA 拷贝/mL)1,2 。在大约10%的肾脏移植接受者中,BK-多瘤病毒(BKPyV)的重新激活导致多瘤病毒相关肾病(PyVAN),高达80%与肾异体移植失败的高风险相关3,4.由于没有经过批准的抗病毒药物,目前的治疗是基于减少免疫抑制。有趣的是,mTOR抑制剂似乎具有抗病毒作用;因此,将免疫抑制疗法切换到基于mTOR的免疫抑制可能是防止BKPyV病毒血症5、6、7进展的替代方法。然而,基于mTOR的抗病毒机制目前仍不完全了解。因此,需要测量潜在抗病毒药物在临床相关浓度中的影响的方法。
BKPyV的循环基因组包括大约5 kb,包含一个非编码控制区域(NCCR),作为复制的起源,并同时作为一个双向启动子,推动早期和晚期相mRNA转录的表达。由于自发发生的NCCR-重排、删除和复制在致病性BKPyV8中发现,并且明显累积在PVAN 5,9患者中,原型(wt)和重新排列 (rr) NCCR 活动有助于描述病毒复制性适应性。
如图1所概述,该协议描述了一种常用的方法,通过量化从报告质粒5表达的两种荧光团和eGFP的荧光来测量BKPyV NCCR转录活性。 9,10,11.该程序在SV40大T抗原(lTAg)的存在下进行,它允许分析潜在的抗病毒药物对NCCR活性的早期和晚期的影响5。该测定进一步分析了重组对NCCR活动的影响,并与wt-NCCRs5、9进行了比较。记者将SV40晚聚光信号下行到每个荧光光片开放读取帧的下游,以确保分别对tdTomato和eGFP的两种转录本进行可比和高效的处理。与基于qRT-PCR的方法5,12相比,这种基于FACS的方法代表了低成本和高吞吐量兼容的替代方法,因为没有复杂的提取协议,感染细胞培养,没有昂贵的需要用于免疫荧光染色的抗体。此外,由于通过流式细胞测定分析定义的荧光细胞量,因此也可以定量分析细胞周期抑制剂。
Protocol
本议定书遵循杜伊斯堡-埃森大学医学院伦理委员会(14-6028-BO)批准的人类研究准则。 1. 采集血液或尿液样本,分离多瘤病毒DNA 在EDTA管中采集至少3 mL的血液,或在采集管中收集尿液。 将样品在2,500 x g下离心15分钟。如果需要,将等离子移液器放入新管中,将等离子样品储存在 4°C 下数天,或在 -20°C 冷冻,以延长存储时间。 将40μL蛋白酶K制备到1.5 mL微离心?…
Representative Results
在具有代表性的实验中,通过流式细胞测定测量了BK-多瘤病毒非编码控制区驱动的转录活性。此外,一种mTOR抑制剂,可用于BKPyV重新激活后治疗患者,被测试为抑制病毒早期基因表达。为此,使用双荧光-报告器测定,如之前公布的5。实验设置的总体工作流程方案在F igure 1中进行了说明。首先,根据机构伦理委员会批准的人类研究指南,从?…
Discussion
本文提出了一种常用的方法,用于分析BKPyV非编码控制区域(NCCR)驱动的早期和后期启动器活动。NCCR活性可以同时测量,不需要转染细胞的溶解。此外,可以分析相对大量的细胞,并且不需要共同转染额外的标记,以使荧光值正常化。
此方法的一个关键部分是,克隆的 NCCR 应包含与阿普利森测序所识别的完全相同的序列,该序列对应于患者血浆中存在的大多数变体。为了获得准确的结果?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢芭芭拉·布莱克曼的出色技术援助。这些研究得到了杜伊斯堡大学埃森医学院的IFORES项目以及鲁尔大学和默卡托研究中心(MERCUR)的RIMUR项目的支持。作者感谢约尔根-曼乔特-斯蒂夫通博士奖学金的海伦·塞尔兹尼格和不断的支持。患者材料的收集和使用已获得杜伊斯堡-埃森大学医学院伦理委员会的批准(14-6028-BO)。
Materials
| 100 bp ladder | NEB | N3231 | Any ladder with a range up to 1 kb can be substituted |
| 2-log ladder | NEB | N0550 | Any ladder with a range up to 10 kb can be substituted |
| Agar-Agar, Kobe I | Carl Roth | 5210.3 | |
| AgeI-HF | NEB | R3552 | |
| Ampicillin Natriumsalz Cellpure | Carl Roth | HP62.1 | |
| Aqua ad iniectabilia | Bbraun | 2351744 | can be substituted by any manufacturer |
| BD FACSCanto™ II | BD Biosciences | ||
| BD FACSDiva | BD Biosciences | ||
| DAPI | Sigma | 10236276001 | |
| DFC450C camera module | Leica | Any camera can be used | |
| DMEM | Gibco | 41966-029 | can be substituted by any manufacturer |
| DMIL LED microscope | Leica | Any fluorescence microscope can be used | |
| DNA Blood Mini Kit | Qiagen | 51104 | can be substituted by any manufacturer |
| E.coli DH5alpha Competent Cells | Thermo Scientific | 18258012 | |
| FBS Superior | MerckMillipore | 50615 | Any FBS can be used |
| FlowJo v10.5.3 | FlowJo, LLC | Any flow cytometry software FBS can be used | |
| Gel Loading Dye, Purple (6X) | NEB | B7024 | Any 6x loading dye can be substituted |
| HEK293T cells | ATCC | 11268 | These cells constitutively express the simian virus 40 (SV40) large T antigen, and clone 17 was selected specifically for its high transfectability. |
| HERAcell® 240i CO2 Incubator | Thermo Scientific | can be substituted by any manufacturer | |
| HotStar PCR kit | Qiagen | 203203 | |
| Intas Gel documentation system | Intas | Any visualisation system for stained DNA containing agarose gels can be used | |
| Low Melt Agarose | Biozym | 850081 | can be substituted by any manufacturer |
| Opti-MEM | Invitrogen | 31985070 | can be substituted by any manufacturer |
| pBKV (34-2) | ATCC | 45025 | Plasmid harboring the full-length genome of BKPyV strain Dunlop; was used as a positive control; DNA Seq. Acc.: KP412983 |
| PBS | Gibco | 14190-136 | |
| PCR Cycler MJ Mini 48-Well Personal Cycler | Bio-Rad | discontinued product | Any thermocycler can be used |
| PCR Nucleotide Mix, 10 mM | Promega | #C1145 | can be substituted by any manufacturer |
| PCR1 and PCR2 Primers | metabion | not applicable | Desalted. Dilute to (10 µM) with PCR grade water |
| PenStrep (100x) | Gibco | 15140-122 | can be substituted by any manufacturer |
| Roti®-GelStain | Carl Roth | 3865 | A fluorescence based stain for measuring dsDNA concentration |
| SpeI-HF | NEB | R3133 | |
| T4-DNA Ligase | NEB | M0202 | |
| TransIT LT1 | Mirus | MIR2300 | |
| Trypsin 0.05% – EDTA | Gibco | 25300-054 | can be substituted by any manufacturer |
| ZymoPURE II Plasmid Kit | Zymo | D4201 | can be substituted by any manufacturer |
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Citer Cet Article
Korth, J., Sertznig, H., Moyrer, S., Doevelaar, A. A. N., Westhoff, T. H., Babel, N., Witzke, O., Kribben, A., Dittmer, U., Widera, M. Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry. J. Vis. Exp. (149), e59755, doi:10.3791/59755 (2019).