生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em>上皮细胞
Summary
一种基于梯度改性密度离心方法被利用来上皮细胞从扇头蜱microplus肠组织隔离。生物素化表面结合蛋白，并通过链霉抗生物素蛋白磁珠纯化，用于下游应用。
Abstract
Rhipicephalus microplus – 牛蜱 – 是对家畜作为几种病原体的载体的经济影响方面最重要的外寄生物。致力于牛蜱控制以减少其有害影响，重点是发现位于蜱肠上皮细胞表面的疫苗候选物质，例如BM86。目前的研究重点是利用cDNA和基因组文库筛选其他候选疫苗。蜱肠细胞的分离是调查蜱肠细胞膜上表面蛋白质组成的重要优势。本文构建了一种用于分离上皮细胞的新颖可行的方法，从半加入的微量元素的蜱肠内含量。该方案利用TCEP和EDTA从上皮支持组织和不连续密度离心分离器中释放上皮细胞以将上皮细胞与其他细胞类型分开。细胞表面蛋白质生物素化并从蜱肠上皮细胞中分离，使用链霉抗生物素蛋白连接的磁珠，允许在FACS或LC-MS / MS分析中的下游应用。
Introduction
扇头蜱microplus，牛蜱，是在对养牛业热带和亚热带地区，因为它矢量牛蜱传热（巴贝虫病），微粒孢子虫病和马焦虫病1，2，3，4经济影响方面最显著外寄生物。努力致力于牛蜱控制，减少有害影响，然而常规方法如使用化学杀螨剂具有隐含的缺点，例如在牛奶和肉中存在化学残留物，以及化学抗性蜱的流行率增加5，6，7。因此，研究了替代蜱控制方法的开发，例如使用天然抗性牛，生物防治（生物杀虫剂）和疫苗分级表4，5，6，7，8，9。
在追求能够被用作候选疫苗的蛋白质时，目前的研究集中在滴滴肠上。中肠壁由沉积在薄基底层上的单层上皮细胞构成，基底层的外侧形成肌肉网络。光和电子显微镜观察表明，中肠由三种类型的细胞组成：储备（未分化），分泌和消化。细胞类型的数量根据生理期有很大差异。分泌和消化细胞都从储备细胞18，19，20发起。
cDNA文库的构建检查蜱肠道的组合物导致了抗原性蛋白质，如Bm86进行的鉴定，作为可能的候选疫苗2，3，4。糖蛋白Bm86位于蜱肠细胞的表面，并在接种疫苗的牛中诱导针对牛蜱（ R. microplus ）的保护性免疫应答。被免疫的宿主产生的抗Bm86IgG被蜱摄入，在蜱肠细胞的表面上识别该抗原，随后扰乱了肠道组织的功能和完整性。基于Bm86抗原的疫苗通过减少雌性哺乳动物的数量，重量和繁殖能力，显示了R. microplus和Rhipicephalus annulatus的有效控制，从而导致后续蜱代4发生减少的幼虫侵袭。然而，基于Bm86的疫苗对于所有的蜱细胞阶段都没有效果表现出对R. microplus的一些地理株不能令人满意的功效，因此在肉牛和奶牛行业都很差采用这些疫苗2,4。
从蜱肠分离上皮细胞的能力是一个重要的创新，这将使研究的进展能够确定不同环境条件下的蛋白质膜组成，包括形态学和生理学。本文所述的方法利用螯合剂乙二胺四乙酸（EDTA）和还原剂三（2-羧乙基）膦（TCEP）从其上皮支持组织10释放上皮。通过振荡机械破坏组织，然后在Percoll中进行不连续梯度离心，回收上皮细胞。本文介绍了一种可行和新颖的用于分离蜱肠道外切的技术血细胞。随后在下游应用如FACS和/或LC-MS / MS分析中分析从这些上皮细胞表面分离的生物素化的细胞表面蛋白质。
Protocol
1.从R. microplus肠上皮的解剖在实验当天从牛收集半加成蜱。从主机取出后24小时内解剖。 在92 mm x 16 mm培养皿的底部附上一条胶带。在胶带上加一滴超级胶水。将蜱，腹侧放在超级胶上，让其干燥2分钟。 将100 mL磷酸盐缓冲盐水（PBS）倒入培养皿中，或直到蜱被完全浸没。 使用大小11的手术刀，从眼睛的顶部切割到底部的festoons，在刻度的两边。 ?…
Representative Results
根据图1所示的原理图，从R. microplus的肠组织中分离上皮细胞。 使用该方案制备的蜱肠上皮细胞的代表性荧光显微镜图像显示在图2A中 和2B。 由于细胞分离是在半富集的R. microplus上进行的，细胞在整个样品中表现为单数，球形，光滑的表面…
Discussion
牛蜱侵染构成了养牛业在世界的热带和亚热带地区的一个主要问题，与控制时使用杀螨剂1，4依赖的最常用的方法。 Bm86进行先前蜱肠上皮表面作为对抗R. microplus侵染10保护性抗原，以有限的成功作为疫苗策略内识别由于Bm86进行地理序列变异和定期升压4的要求。
以前的出版物聚焦在上皮隔?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者希望感谢澳大利亚昆士兰州农业和渔业部门提供用于本研究的Rhipicephalus microplus蜱，以及Lucas Karbanowicz协助视频拍摄。
Materials
| 0.4% Trypan Blue | ThermoFisher Scientific | 15250061 | |
| 1.5 mL microcentrifuge tube | Eppendorf | 3322 | |
| 100mM Carbonate Buffer | 3.03 g Na2CO3, 6.0 g NaHCO3 1000 ml distilled water pH 9.6 | ||
| 16 mL centrifuge tubes with sealing cap | Thermo Scientific | 3138-0016 | Cool in ice prior to gradient |
| 250 µM cell strainer | Thermo Fisher | 87791 | |
| 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA | Sigma | T0440 | Stored at 4C |
| 30% Hydrogen Peroxide | Labscene | BSPA5.500 | |
| 4-20% Tris-MOPS Gel | Gen Script | M42015 | |
| 4-Chloro-1-naphthol tablet | Sigma-Aldrich | C6788 | |
| 50 mL Falcon Tube | Corning Blue | 30 x 115mm style. Polyproplyene conical tube. | |
| 70 µM cell strainer | BD Falcon | 352350 | |
| AP15 filter paper | Millipore | AO1504200 | |
| Biotin (Type A) Conjugation Kit | Abcam | Ab102865 | |
| Dissection microscope | Olympus | SZX7 | |
| DP Manager | Olympus | 2.2.1.195 | Cell imagery software |
| Duct Tape | Home Handyman | 48mm x 25mm Duct Tape | |
| Dulbecco’s Modified Eagle Medium | Gibco | 11995-065 | DMEM – ice cold for protocol |
| EDTA | Amresco | 0105-500G | |
| F96 Maxisorp Immuno Plate | Nunc | 439454 | |
| Fetal Bovine Serum | Sigma-Aldrich | 12003C | FCS |
| Fluorescence microscope | Olympus | BX51 | |
| Fluoroshield with DAPI | Sigma-Aldrich | F6057-20ML | DAPI |
| Forceps | Dumont | #9 Dumont – Switerzland | |
| Glycerol | Sigma-Aldrich | G5516 | Glycerol for molecular biology >99% |
| Glycine | Sigma-Aldrich | 410225 | |
| Hand-Held Counter | Officeworks | JA0376230 | |
| Hank’s Balanced Salt Solution | Sigma Life Sciences | H9394 | HBSS – ice cold for protocol |
| Hemacytometer | Optik Lakor | – | – |
| L-Glutathione oxidized | Sigma-Aldrich | G4376 | |
| Magnetic Separation Stand | Novagen | – | 4-Tube Magnetic Separation Rack |
| Methanol | Sigma-Aldrich | 179337 | |
| Milli-Q Water | Millipore | ZRXQ003WW | Integral Water Purification System for Ultrapure Water |
| Nitrocellulose Membrane | Life Sciences | 66485 | 30cm x 3M pure nitrocellulose membrane |
| PageRuler Prestained protein Ladder | Thermo-Fisher | SM0671 | |
| PBS | 1.16 g Na2HPO4, 0.1 g KCl, 0.1 g K3PO4, 4.0 g NaCl (500 ml distilled water) pH 7.4 | ||
| Percoll | Sigma-Aldrich | P1644-500ML | |
| Peristaltic Pump | Masterflex | 7518-10 | |
| Phosphoric Acid | Sigma-Aldrich | P6560 | |
| Pierce Protein-Free T20 PBS Blocking Buffer | Thermo-Scientific | 37573 | Stored at 4C. Blocking Buffer |
| Protease Inhibitor Cocktail | Sigma-Aldrich | P8215-5ML | PIC – stored at -20 °C |
| Quick Start Bradford Dye Reagent 1x | Biorad | 500-0205 | For Bradford Assay |
| Quick Start BSA Standards | Biorad | 500-0207 | BSA standards for Bradford Assay |
| Scalpel | Lab. Co | Size 11 Scalpel | |
| SilverQuest TM Staining Kit | Invitrogen | LC6070 | |
| Simply Blue TM Safe Stain | Invitrogen | LC6060 | |
| Sorvall C6+ Ultracentrifuge | Thermo Scientific | 46910 | |
| Streptavidin (HRP) | Abcam | AB7403 | |
| Streptavidin Magnetic Beads | New England Biolabs | S1420S | |
| Super Glue – Ultra Fast Mini | UHU | UHU Super Glue 1mg. Ultra Fast mini | |
| Table-top Centrifuge | Eppendorf | 22331 | |
| TCEP | Thermo Fisher | 20490 | |
| Triton X-100 | Biorad | 161-0407 | |
| Tween-20 | Sigma | P2287-500ML | |
| Vortex Mixer | Ratek | VM1 | |
| Water Bath | Grant | GD100 |
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Cite This Article
Karbanowicz, T. P., Lew-Tabor, A., Rodriguez Valle, M. Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells. J. Vis. Exp. (125), e55747, doi:10.3791/55747 (2017).