Summary

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

Published: October 27, 2016
doi:

Summary

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Abstract

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

Introduction

MS是人类疾病的特点是慢性炎症和这被认为是由针对髓鞘自身免疫应答被驱动的中枢神经系统的神经变性。髓鞘和轴突随时间的流失导致认知和运动功能1逐渐下降。 “实验性自身免疫性脑脊髓炎”是用于针对CNS髓磷脂自身免疫性疾病的动物模型的总称。像人类的MS,EAE特征通常为CNS的免疫细胞浸润,并且在某些情况下,脱髓鞘2。然而,向其中任何给定的EAE模型类似于人的MS部分的程度取决于所使用的种或菌株和在底层的抗髓鞘自身免疫反应的复杂性。

抗髓鞘自身免疫可以通过实验诱导几种方式,但是目前使用的最常见的方法是将免疫小鼠用氨基酸模仿免疫的CD4的短肽<s了髓鞘蛋白> + T细胞表位。这表示以诱导致病响应的最低要求。可能是最常见的这些是从髓鞘少突胶质糖蛋白(MOG 35-55)衍生的21氨基酸肽,它用于以诱导EAE在C57BL / 6小鼠3。然而,对于一些实验目的,希望或甚至必需的具有较大蛋白抗原和确实有一些优点这个过短肽免疫来免疫。第一,因为MHC限制的,短肽通常只有在菌株的一个非常有限的范围内有效,而代表任一整蛋白或特定域较大蛋白抗原可在不同的物种中正常处理以便呈现在多个近交小鼠品系或甚4。第二,较大的蛋白抗原能够诱导结合多种类型的淋巴细胞的抗原识别,更复杂的免疫反应,而不是limitin的15μg抗原识别到CD4 + T细胞。例如,通过其B细胞受体(BCR)的B细胞与整体,而不是处理的蛋白直接相互作用。我们和其他人已经表明由MOG 35-55免疫激活不承认MOG蛋白5 B细胞。由于B细胞最近证明在人MS 6发挥致病作用,即结合B细胞在自身免疫性病理学的EAE模型是越来越重要。

尽管使用较大的蛋白抗原以诱导EAE的优点,仍存在这样的蛋白质的一些可商购的来源。实际上,虽然短肽像MOG 35-55可以非常快速地并以相对低的成本来合成MOG蛋白的商业选择是有限的,成本基本上更购买。然而,存在可用于研究团体产生MOG胞外结构域的几个表达载体(MOG 1-125)本身。 HoweveR,所有我们在文献中已经确定了表达系统是基于至今已换成更高效表达系统7的旧技术。此外,大多数是基于大鼠或人MOG 8。用于在小鼠中自身免疫的一些调查,根据鼠标MOG自身抗原的抗原是优选的。最后,商业或作为表达载体,我们已经确定了所有基于MOG蛋白,是含有额外的氨基酸的MOG 1-125基的融合蛋白。这些措施包括净化,通常其他序列标签为好,这有很多的功能,我们无法辨认。

为了解决这些局限性,我们产生基于稠合到含有硫氧还蛋白打击MOG蛋白5的已知的不溶性的标记的小鼠MOG胞外结构域的新型融合蛋白。标签序列还包含用于净化和TEV蛋白酶CLE一个序列的6xHisavage站点,允许完全去除所有的标签序列的,如果需要的话。这是我们都知道的,生成纯MOG 1-125蛋白质的唯一方法。为了便于生产大量蛋白质的MOG 1-125序列用于细菌表达密码子优化和MOG 标签融合蛋白插入的pET-32表达系统。在这里,我们详细描述了该协议生产和净化MOG 标记蛋白,而纯MOG-125,使用现有的大多数实验室免疫学非专用设备。

Protocol

1.蛋白质感应注意:在以下步骤,BL21 大肠杆菌细菌转化含用于MOG 标签融合蛋白的序列的的pET-32载体(见参考图5和图1)中生长到高密度,然后被诱导表达的MOG 标记蛋白。 图2所示为整体时间表-注意,天都是近似值和备用停机点在协议中注明。如果与纯化的pET-32 MOG 标签载体DNA开始,这将是必要的,以化学它转变…

Representative Results

一旦纯化完成后,收集在步骤1.4,2.1,3.4的样品,并从步骤6.4最终产物应在蛋白凝胶( 图3A)中运行。 MOG 标签应首先出现当T O / N样品中的31.86 kDa带,但不是T 0,并应在最终纯产物的唯一频带。为了测试MOG 标记蛋白是否已经正确折叠时,MOG 标签蛋白可用于通过FACS标记MOG蛋白特异性B细胞。通过用1标记小鼠淋巴细胞:MO…

Discussion

这里,我们已经描述了协议用于生产MOG 标记蛋白以及如何产生来自MOG 标签蛋白质纯MOG 1-125的。这个协议是基于两个标准His标签的蛋白质的纯化方法,以及用于一个较旧的基于MOG蛋白15的产生先前描述的方案。虽然没有在这里描述的,MOG 标签蛋白的主要用途是通过与蛋白质抗原免疫以诱导EAE。描述的EAE是如何在小鼠,这是与MOG 标签蛋白兼容诱导的?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by a grant from the Multiple Sclerosis Society of Canada. RWJ is the recipient of the Waugh Family MS Society of Canada Doctoral Studentship Award.

Materials

BL21 E.coli– pet32-MOGtag Kerfoot lab These bacteria are required to make the MOGtag protein. Glycerol stocks of these bacteria are available upon request.
LB broth miller Bioshop LBL407.1
Ampicillin bio basic AB0028 Reconsititute the powder into 50% ethanol/ 50% H2O at 100 mg/ml. Store at -20 °C.
IPTG Bioshop IPT002.5 Reconsititute the powder into H2O at 1M and store at -20 °C.
Chicken-egg lysozyme Bioshop LYS702.10 Reconstitute in H2O at 50 mg/ml and store at -80 °C.
Triton-X100 Sigma T-8532
Phosphate buffered saline life technologies 20012-027 Commercial phosphate buffered saline is not required, any standard lab made phosphate buffered saline is sufficient.
Sodium chloride Bioshop SOD004.1
Tris-HCl Bioshop TRS002.1
Imidazole Bioshop IMD508.100
Guanidine-HCl Sigma G3272 The quality must be greater than 98% purity.
0.5M EDTA bioshop EDT111.500
Nickel (II) sulfate Bioshop NIC700.500
His bind resin EMD Millipore 69670-3 Store in 20% ethanol 80% H2O at 4 °C
Anhydrous ethanol Commercial Alcohols P016EAAN Dilute with water as needed.
Glacial acetic acid Bioshop ACE222.1
Sodium acetate trihydrate Bioshop SAA305.500
bovine serum albumin standard bio-rad 500-0206
Bio-rad protein assay dye reagent concentrate bio-rad 500-0006
Ethylenediamine tetraacetic acid, disodium salt dihydrate Fisher scientific BP120-500
Tris-base Bioshop TRS001.1
7000 MW Snakeskin dialysis tubing Thermoscientific 68700
2-mercaptoethanol Sigma M3148-25ml This reagent should not be handled outside of a fume hood.
AcTEV protease lifetechnologies 12575-015 Producing your own TEV protease can be accomplished using (https://www.addgene.org/8827/) and purified as in reference 17
Polyethyleneglycol 3350 Bioshop PEG335.1
polyethyleneglycol 8000 Bioshop PEG800.1
Nunc MaxiSorp flat-bottom 96 well plate ebioscience 44-2404-21
Sonicator Fisher scientific FB-120-110
Eon microplate spectrometer Biotek 11-120-611 This equipment uses the Gen5 data analysis software.
Gen5 data analysis software BioTek
sodium dodecyl sulphate Bioshop SDS001
bromophenol blue Bioshop BRO777
Glycerol Bioshop GLY001
Protein desalting columns Thermoscientific 89849
Glycine Bioshop GLN001
precast 12% polyacrylamide gel bio-rad 456-1045
Rapid stain reagent EMD Millipore 553215
Gel dock EZ imager bio-rad 1708270
White Light Sample Tray bio-rad 1708272  Used along with gel dock EZ imager for coomassie blue stains
Protein ladder bio-rad 1610375

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Cite This Article
Jain, R. W., Dang, A. K., Kerfoot, S. M. Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies. J. Vis. Exp. (116), e54727, doi:10.3791/54727 (2016).

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