Name: simple and efficient production and purification of mouse myelin oligodendrocyte glycoprotein for experimental autoimmune encephalomyelitis studies
Uploaded: 2016-10-27T14:00:00
Description: Watch this Scientific Journal Video about Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies at JoVE.co

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.

1. 단백질 유도 참고 : 다음 단계에서는 BL21 대장균 박테리아 MOG 태그 융합 단백질에 대한 서열을 포함하는 애완 동물-32 벡터로 형질 전환 된 고밀도로 성장하고 MOG 태그 단백질을 발현하도록 유도된다 (문헌 5 및도 1 참조) . 일 대략적인 대체 정지 점 프로토콜에 설명되어 있습니다 참고 - 전체 타임 라인 그림 2를 참조하...

<ol>
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여기서는 MOG 태그 단백질 및 방법 MOG 태그 단백질 순수한 MOG 1-125을 생성하는 생산을위한 프로토콜을 기술 하였다. 이 프로토콜은 표준 그의 태그 기반의 단백질 정제 방법뿐만 아니라 이전 MOG 단백질 계 (15)의 생성을위한 전술 한 두 프로토콜에 기초한다. 그것은 여기에서 설명되지 않지만, MOG 태그 단백질의 주 사용은 단백질 항원을 면역 EAE를 유도하는 ?...

MS 만성 염증과 미엘린 향하는자가 면역 반응에 의해 구동되는 것으로 생각되는 CNS의 신경 퇴행을 특징으로하는 인간의 질병이다. 시간이 지남에 따라 수초와 축색 돌기의 손실은인지 및 모터 기능을 하나의 점진적 감소가 발생. &quot;실험자가 면역 뇌척수염은&quot;중추 신경계의 수초 지향자가 면역 질환의 동물 모델에 대한 포괄적 인 용어이다. 인간의 MS와 마찬가지로 EAE는 일반적으로 어떤 경우에는 CNS의 면역 세포 침윤을 특징으로하고,이 탈수 초화. 그러나, 임의의 주어진 EAE 모델은 부분적으로 인간의 MS와 유사한 과정에 사용되는 화학 종 또는 균주 및 하부 반 미엘린 면역 반응의 복잡성에 좌우된다. 안티 미엘린 실험적자가 면역은 여러 가지 방법으로 유도하지만, 현재 사용되는 가장 일반적인 방법은 면역 CD4 흉내 아미노산의 짧은 펩타이드 마우스 면역화 될 수있다 <s미엘린 단백질&gt; + T 세포 에피토프입니다. 이 병원성 반응을 유도 할 수있는 최소 요구 사항을 나타냅니다. 아마도 이들 중 가장 일반적인 C57BL / 6 마우스 3 EAE를 유도하기 위해 사용된다 (MOG 35-55) 미엘린 희소 돌기 아교 세포의 당 단백질에서 유래하는 21 개의 아미노산 펩타이드이다. 그러나 일부 실험 목적이 큰 단백질 항원 실제로 짧은 펩타이드와 예방 접종을 통해이 몇 가지 장점이와 예방 접종하는 것이 바람직 심지어 필요가있다. 전체 단백질 또는 특정 도메인의 어느 쪽인지를 표현하는 더 큰 단백질 항원이 다른 종에서도 여러 근친 마우스 변종에 프리젠 테이션을 위해 정상적으로 처리하거나 할 수 있지만 먼저, MHC의 제한 때문에, 짧은 펩티드는, 일반적으로 균주의 매우 제한된 범위에서만 유효하다 4. 둘째로, 더 큰 단백질 항원은 항원 인식 림프구 종 이상을 포함하는보다 복잡한 면역 반응을 유도하기보다는 limitin 할CD4 + T 세포에 항원 g 인식. 예를 들어, 그들의 B 세포 수용체 (BCR)을 통해 B 세포는 전체 단백질보다는 처리와 직접 상호 작용한다. 우리와 다른 MOG 단백질 (5)을 인식하지 못하는 MOG 35-55 예방 접종에 의해 활성화하는 B 세포를 보여 주었다. B 세포는 인간 최근 MS 6에서 병원성 역할을하는 것으로 입증 되었기 때문에,자가 면역 병리에서 B 세포를 포함 EAE 모델은 점점 중요하다. EAE를 유도하는 큰 단백질 항원을 사용하는 장점에도 불구하고, 단백질에 대한 몇 시판 소스가 남아있다. MOG 35-55 같은 짧은 펩티드가 매우 빠르고 상대적으로 낮은 비용으로 합성 할 수 있지만 실제로, MOG 단백질 상업 옵션을 구입 제한 및 비용 실질적으로 더 있습니다. 그럼에도 불구하고, MOG 세포 외 도메인을 생성하는 연구 그룹에 사용할 수있는 여러 가지 발현 벡터 (MOG 1-125) 자신이있다. HoweveR, 우리는 문헌에서 확인 하였다 발현 시스템의 모든 사람보다 효율적인 발현 시스템 (7)로 대체 한 이전 기술에 기초한다. 또한, 대부분의 쥐 또는 인간의 MOG 8을 기반으로합니다. 마우스에서자가 면역 반응의 일부 수사 마우스 MOG의자가 항원에 기초하여 항원이 바람직하다. 마지막으로, 상업적 또는 발현 벡터와 같은 하나 우리가 식별 된 모든 MOG 계의 단백질은 상기 MOG 1-125 기재에 추가적인 아미노산을 포함하는 융합 단백질이다. 다음은 우리가 식별 할 수 없습니다 많은 기능이있는뿐만 아니라 정화 및 일반적으로 다른 시퀀스에 대한 태그를 포함한다. 이러한 한계를 해결하기 위해, 우리는 MOG 단백질 5의 공지 불용성 싸우는 오레 독신을 포함하는 태그에 융합 된 마우스 MOG 외 도메인에 기초하여 신규 한 융합 단백질을 생성. 태그 시퀀스는 정화와 TEV 프로테아제 CLE에 대한 6xHis 시퀀스를 포함원하는 경우, 모든 태그 서열의 완전한 제거를 허용 avage 사이트. 이것은 우리가 순수 MOG 1-125 단백질을 생성 할 알고있는 유일한 방법입니다. 단백질의 대량 생산을 용이하게하기 위해, MOG 1-125 서열은 박테리아 발현 코돈 - 최적화하고, MOG 태그 융합 단백질 애완-32 발현 시스템 내로 삽입되었다. 여기서는 가장 면역학 연구실 사용할 비 특수 장비를 사용하여 생산 MOG 태그 단백질 순수 MOG 1-125를 정화 상세히 프로토콜을 설명한다.

<table border="0" cellpadding="0" cellspacing="0" width="1434">
	<tbody>
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			<td height="25" style="height:25px;width:375px;">BL21 E.coli- pet32-MOGtag</td>
			<td style="width:139px;">Kerfoot lab</td>
			<td style="width:119px;"></td>
			<td style="width:803px;">These bacteria are required to make the MOGtag protein. Glycerol stocks of these bacteria are available upon request.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LB broth miller</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LBL407.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Ampicillin</td>
			<td style="width:139px;">bio basic</td>
			<td style="width:119px;">AB0028</td>
			<td>Reconsititute the powder into 50% ethanol/ 50% H2O at 100 mg/ml. Store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">IPTG</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IPT002.5</td>
			<td>Reconsititute the powder into H2O at 1M and store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Chicken-egg lysozyme</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LYS702.10</td>
			<td>Reconstitute in H2O at 50 mg/ml and store at -80 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Triton-X100</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">T-8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Phosphate buffered saline</td>
			<td style="width:139px;">life technologies</td>
			<td style="width:119px;">20012-027</td>
			<td>Commercial phosphate buffered saline is not required, any standard lab made phosphate buffered saline is sufficient.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium chloride</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SOD004.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-HCl</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS002.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Imidazole</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IMD508.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Guanidine-HCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">G3272</td>
			<td>The quality must be greater than 98% purity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">0.5M EDTA</td>
			<td style="width:139px;">bioshop</td>
			<td style="width:119px;">EDT111.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nickel (II) sulfate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">NIC700.500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">His bind resin</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">69670-3</td>
			<td>Store in 20% ethanol 80% H2O at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Anhydrous ethanol</td>
			<td style="width:139px;">Commercial Alcohols</td>
			<td style="width:119px;">P016EAAN</td>
			<td>Dilute with water as needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glacial acetic acid</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">ACE222.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium acetate trihydrate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SAA305.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bovine serum albumin standard</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Bio-rad protein assay dye reagent concentrate</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Ethylenediamine tetraacetic acid, disodium salt dihydrate</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-base</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">7000 MW Snakeskin dialysis tubing</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">68700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">2-mercaptoethanol</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">M3148-25ml</td>
			<td>This reagent should not be handled outside of a fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">AcTEV protease</td>
			<td style="width:139px;">lifetechnologies</td>
			<td style="width:119px;">12575-015</td>
			<td>Producing your own TEV protease can be accomplished using (https://www.addgene.org/8827/) and purified as in reference 17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Polyethyleneglycol 3350</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG335.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">polyethyleneglycol 8000</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG800.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nunc MaxiSorp flat-bottom 96 well plate</td>
			<td style="width:139px;">ebioscience</td>
			<td style="width:119px;">44-2404-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sonicator</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">FB-120-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Eon microplate spectrometer</td>
			<td style="width:139px;">Biotek</td>
			<td style="width:119px;">11-120-611</td>
			<td>This equipment uses the Gen5 data analysis software.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gen5 data analysis software</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">sodium dodecyl sulphate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SDS001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bromophenol blue</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">BRO777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycerol</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLY001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein desalting columns</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">89849</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycine</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLN001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">precast 12% polyacrylamide gel</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">456-1045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rapid stain reagent</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">553215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gel dock EZ imager</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">White Light Sample Tray</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708272&#160;</td>
			<td>Used along with gel dock EZ imager for coomassie blue stains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein ladder</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1610375</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple and efficient production and purification of mouse myelin oligodendrocyte glycoprotein for experimental autoimmune encephalomyelitis studies

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.

정화가 완료되면, 샘플 단계 1.4, 2.1, 3.4에서 수집하고, 단계 6.4의 최종 생성물은 단백질 겔 (도 3A)에서 실행되어야한다. MOG 태그 먼저 T O / N 샘플에서 31.86 kDa의 밴드로 나타날 수 있지만 0 T, 최종 순수한 생성물의 유일한 밴드되어야한다. 모그 태그 단백질이 올바르게 접혀 여부를 테스트하기 위해, MOG 태그 단백질 FACS?...

Watch this Scientific Journal Video about Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies at JoVE.co

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

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.

실험자가 면역 뇌척수염 연구 간단하고 효율적인 생산 및 마우스 미엘린 희소 돌기 아교 세포 당 단백질의 정제

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Research

JoVE Journal

Immunology and Infection

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

실험 면역 Hypophysitis의 유도를위한 마우스 뇌하수체 Immunogen의 준비

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

연구

면역학 및 감염병학

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

입양 이전 및 Antigenic 챌린지에 의해 실험적 면역 Encephalomyelitis (EAE) 모성 마우스 변종에 응답하지 않는 문제를 극복

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

수초 희소 돌기 아교 세포 당 단백질 (MOG<sub&gt; 35-55</sub&gt;) C57BL / 6 쥐에 실험자가 면역 뇌척수염 (EAE) 유도

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

다양한 조직에서 면역 세포의 질병에 의존하는 유통의 마우스 및 평가에 실험자가 면역 뇌척수염의 유도

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

Vivo에서 실험적인 자기 면역 뇌 중 Neurovascular 단위의 내 피와 폐해 장벽의 장애 시각화

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

실험적인 자가면역 뇌척수염 중 중추신경계에서 림프구 침투의 흐름 세포 분석

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

실험적 자가면역 포도막염: 안구내 염증성 마우스 모델

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

체외 및 생체 내 연구를 위한 마우스 불변 천연 킬러 T 세포의 정화 및 확장

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

실험적 자가면역 뇌척수염 유도를 위한 항원/보조 에멀젼을 제조하기 위한 빠르고 간단하며 표준화된 균질화 방법

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely ...

Immunogenicity Enhancement by Mycobacterium paratuberculosis in Autoimmune Encephalomyelitis

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis (Video) | JoVE 

실험적 자가면역 뇌척수염 동안 자가항원의 면역원성을 향상시키는 Mycobacterium paratuberculosis 의 보조 활성

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...