肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定
Summary
Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.
Abstract
抗原表位的免疫系统的识别允许用于中和可便于疫苗和肽类药物的发展抗体的保护机制的理解。肽扫描是直截了当映射由单克隆抗体（mAb）识别的线性表位的简单而有效的方法。在这里，作者提出了涉及连续截断重组蛋白，合成肽设计和斑点杂交使用中和单克隆抗体神经坏死病毒外壳蛋白的抗原识别表位确定方法。此技术依赖于合成肽和单克隆抗体上的聚偏二氟乙烯（PVDF）膜的斑点杂交。由RG-M56单克隆抗体识别的病毒外壳蛋白的最小抗原区可以通过一步一步的收窄修整肽作图到6聚体的肽表位。此外，丙氨酸扫描诱变和残余物分可以进行stitution来表征每个氨基酸残基组成的表位的结合的意义。发现侧翼表位的现场残留发挥肽结构调控关键作用。所识别的表位肽可被用来形成用于X射线衍射研究和功能的竞争，或用于治疗的表位肽 – 抗体复合物的晶体。
Introduction
在免疫系统中，V，D和J片段的重组允许抗体结合至各种抗原，以保护从病原体感染的主机创建互补决定区（CDR）的巨大变化。针对抗原的抗体的中和防御依赖于抗体的CDR和抗原的抗原决定簇之间的空间互补性。因此，这种分子相互作用的理解将有助于预防性疫苗的设计和治疗肽的药物开发。然而，这种中和的相互作用可以都通过从一个单一的抗原多抗原结构域和由抗体的多个的CDR，其结果使表位判定处理更复杂的影响。幸运的是，杂交瘤技术的发展，该熔断器个体抗体产生细胞与骨髓瘤细胞，允许细胞不断地分裂批次秒RETE一个特定抗体称为单克隆抗体（mAb）1。杂交瘤细胞生产这些纯，高亲和力的单克隆抗体绑定到特定抗原的单一抗原域。既定的抗原 – 抗体，几种方法，包括肽扫描的关系，可用于确定使用其相应的单克隆抗体的抗原的表位。在合成肽技术的最新发展使得肽扫描技术更容易，更方便进行。简单地说，一组重叠的合成肽的根据靶抗原序列产生和关联到用于单抗杂交的固体支持膜。肽扫描不仅提供了一种简单的方式来映射抗体结合区，但也有利于氨基酸（AA）的诱变通过残扫描或取代来评估表位肽的每个氨基酸残基和抗体的CDR之间的结合相互作用。
<p c小姑娘="“jove_content”">这里，本研究描述了使用中和单克隆抗体2，3，4中的黄色石斑鱼神经坏死病毒（YGNNV）外壳蛋白的线性表位的有效识别的协议。该协议包括单克隆抗体制备，结构和串联截短重组蛋白的表达，合成的重叠肽的设计，斑点杂交，丙氨酸扫描，和替代诱变。考虑肽合成的高成本，的串行截断所需靶蛋白的重组蛋白的步骤被修改，进行合成肽阵列斑点印迹分析前的抗原区被缩小到约100至200个氨基酸残基。Protocol
Representative Results
Discussion
该协议提供了一个快速，简单的技术来识别单抗公认的线性表位。考虑到肽合成和合成肽的生产效率的成本，病毒外壳蛋白的抗原区由前肽扫描分析表达串联截短重组蛋白减少。因此，使用可靠的和有效的大肠杆菌 pET表达系统产生这些串联截短的重组蛋白，与50 10之间的分子量的重组蛋白质kDa的可以容易地通过该系统中表达。在这种方式中，表位可以容易地缩小到一个更容易管理的100到200?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank Miss Ching-Chun Lin and Miss Diana Lin of the Core Facility of the Institute of Cellular and Organismic Biology (ICOB) of Academia Sinica for offering their expertise on peptide synthesis and DNA sequencing, respectively. This study was supported by Academia Sinica.
Materials
| Hybrid-SFM medium | Gibco | 12045-076 | |
| Dulbeccos's Phophate-Buffered Saline (PBS) | Gibco | 21600-069 | |
| Pfu DNA Polymerase | Thermo Scientific | EP0502 | Including buffers |
| T4 DNA Ligase | Roche | 10799009001 | Including buffers |
| NdeI | New England Biolabs | R0111S | Including buffers |
| XhoI | New England Biolabs | R0146S | Including buffers |
| pET-20b(+) vector | Novagen, Merck Millipore | 69739 | |
| E.coli DH-5α competent cell | RBC Bioscience | RH617 | |
| E.coli BL-21(DE3) competent cell | RBC Bioscience | RH217 | |
| Ampicillin | Amresco | 0339-25G | |
| LB broth | Invitrongen | 12780-052 | |
| Isopropylthio-β-D-thiogalactoside (IPTG) | MDBio, Inc. | 101-367-93-1 | |
| Methanol | Merck Millipore | 106009 | |
| Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20) | J.T.Baker | X251-07 | |
| Dimethyl sulfoxide (DMSO) | Sigma | D2650 | |
| Glycine | Amresco | 0167-5KG | |
| Tris | Affymetrix, USB | 75825 | |
| NaCl | Amresco | 0241-1KG | |
| EDTA | Amresco | 0105-1KG | |
| Glycerol | Amresco | 0854-1L | |
| NaN3 | Sigma | S2002-500G | |
| BCIP/NBT | PerkinElmer | NEL937001PK | |
| Goat Anti-Mouse IgG, Fc fragment antibody | Jackson ImmunoResearch | 115-055-008 | |
| Immobilon-P (Polyvinylidene fluoride, PVDF) | Merck Millipore | IPVH00010 | |
| Protein G Agarose Fast Flow | Merck Millipore | 16-266 | |
| QIAquick PCR Purification kit | Qiagen | 28106 | |
| UVP BioSpectrum 600 Image System | UVP | n/a | |
| VisionWorks LS Analysis Software Ver 6.8 | UVP | n/a | |
| MyCycler thermal cycler | BioRad | 1709713 |
Referências
- Milstein, C., Kohler, G. Clonal variations of myelomatous cells (proceedings). Minerva Med. 68 (50), 3453 (1977).
- Lai, Y. S., et al. In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper Epinephelus awoara (Temminck & Schlegel). J Fish Dis. 24 (4), 237-244 (2001).
- Chen, C. W., Wu, M. S., Huang, Y. J., Cheng, C. A., Chang, C. Y. Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection. PLoS One. 10 (5), 0126121 (2015).
- Lai, Y. -. S., et al. Propagation of yellow grouper nervous necrosis virus (YGNNV) in a new nodavirus-susceptible cell line from yellow grouper, Epinephelus awoara (Temminck & Schlegel), brain tissue. J Fish Dis. 24 (5), 299-309 (2001).
- Lougee, E., Morjaria, S., Shaw, O., Collins, R., Vaughan, R. A new approach to HLA typing designed for solid organ transplantation: epityping and its application to the HLA-A locus. Int J Immunogenet. 40 (6), 445-452 (2013).
- Radulovich, N., Leung, L., Tsao, M. S. Modified gateway system for double shRNA expression and Cre/lox based gene expression. BMC Biotechnol. 11, 24 (2011).
- Froger, A., Hall, J. E. Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp. (6), e253 (2007).
- Pronobis, M. I., Deuitch, N., Peifer, M. The Miraprep: A Protocol that Uses a Miniprep Kit and Provides Maxiprep Yields. PLoS One. 11 (8), e0160509 (2016).
- Metzker, M. L. Emerging technologies in DNA sequencing. Genome Res. 15 (12), 1767-1776 (2005).
- Chiu, C. C., John, J. A., Hseu, T. H., Chang, C. Y. Expression of ayu (Plecoglossus altivelis) Pit-1 in Escherichia coli: its purification and immunohistochemical detection using monoclonal antibody. Protein Expr Purif. 24 (2), 292-301 (2002).
- Atassi, M. Z. Antigenic structures of proteins. Their determination has revealed important aspects of immune recognition and generated strategies for synthetic mimicking of protein binding sites. Eur J Biochem. 145 (1), 1-20 (1984).
- Opuni, K. F., et al. Mass spectrometric epitope mapping. Mass Spectrom Rev. , (2016).
- Chang, C. Y., Chiu, C. C., Christopher John, J. A., Liao, I. C., Leaño, E. M. . The Aquaculture of Groupers. , 207-224 (2008).
- Conti-Tronconi, B. M., et al. Alpha-bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite. Bioquímica. 30 (10), 2575-2584 (1991).
- Briggs, S., Price, M. R., Tendler, S. J. Fine specificity of antibody recognition of carcinoma-associated epithelial mucins: antibody binding to synthetic peptide epitopes. Eur J Cancer. 29 (2), 230-237 (1993).
- Chen, N. C., et al. Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection. PLoS Pathog. 11 (10), e1005203 (2015).
- Badani, H., Garry, R. F., Wimley, W. C. Peptide entry inhibitors of enveloped viruses: The importance of interfacial hydrophobicity. Biochim Biophys Acta. , (2014).
- Qureshi, N. M., Coy, D. H., Garry, R. F., Henderson, L. A. Characterization of a putative cellular receptor for HIV-1 transmembrane glycoprotein using synthetic peptides. AIDS. 4 (6), 553-558 (1990).
