Name: stability and structure of bat major histocompatibility complex class i with heterologous &beta;2-microglobulin
Uploaded: 2021-03-10T13:00:00
Description: Watch this Scientific Journal Video about Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous ÃŽÂ²2-Microglobulin at JoVE.com

本研究由中国南京大学医药生物技术国家重点实验室开放基金资助。KF-GN-201905），中国国家自然科学基金（赠款81971501）。刘晓波得到了国家自然科学基金（81822040）和北京新星科技计划（Z18110006218080）的优秀青年科学家项目的支持。

1. 表达结构的准备
<ol><li>从NCBI数据库中检索MHC I类基因（包括预测基因）的序列。</li>
	<li>从免疫多态性数据库 （IPD） （www.ebi.ac.uk/ipd/mhc） 和 UniProt 数据库 （www.uniprot.org） 检索更高的哺乳动物 MHC I 重链序列。</li>
	<li>要获得可溶性 MHC 复合物，请将序列变异以去除细胞反子和跨膜区域。</li>
	<li>克隆编码蝙蝠Ptal-N*01：01（根银行号）的基因。KT98792919） （残留物 1-277） 和蝙蝠?...

<ol>
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在无法使用同源复合物（如 MHC I 及其配体）时，通过不同分类的异质替代构建混合蛋白复合物是功能和结构研究的常见策略。然而，关于方法和技术的总结有限。在这里，分析了蝙蝠MHC I，Ptal-N*01：01，稳定在b+2米或h+2m的结构。发现与Ptal-N*01：01结合的β 2米键氨基酸在蝙蝠和人类之间保存。经进一步分析，与MHC I H链β 2米结合的关键残留物在哺乳动物中保存，但在...

主要的组织相容性复合物（MHC）存在于所有脊椎动物中，是一组基因，决定细胞介质对传染性病原体的免疫力。MHC类I向CD8+T 细胞表面的T细胞受体（TCR）展示内源肽，如病毒感染时产生的病毒成分，以调节细胞免疫和参与免疫调节1。MHC I 与肽结合的结构研究提供了有关 MHC I 分子的肽结合主题和表现特征的信息，在评估 CD8+ T 细胞免疫反应和疫苗开发中起着至关重要的作用。
自比约克曼等人首次对MHC I分子进行结晶和结构测定以来，MHCI分子的晶体结构分析极大地促进了对肽如何与MHC I分子结合的理解，有助于理解轻链与重链和肽的相互作用。一系列后续研究表明，虽然编码光链的基因与MHC无关，但光链是MHC I分子3、4组位的关键蛋白质。它与多个表面上的 MHC I 类分子的三个域相互作用。当光链缺失时，MHC I类分子不能正确表达在抗原呈现细胞表面，也不能与TCR相互作用以发挥其免疫功能。
MHC I由重链（H链）和轻链（即β 2-微球蛋白（β 2米）组成，通过与合适的肽5结合组装而成。H 链的细胞外部分由 α1、α2 和 α3 域6组成。α1 和 α2 域形成肽结合槽 （PBG）。β 2米链作为MHC I装配综合体的结构子团，稳定了复合物的构象，是MHC I H链折叠7、8、9的分子伴奏。一系列研究表明，MHC I H链来自各种哺乳动物，如蝙蝠（脊柱菌）（Ptal-N*01：01）10，恒河猴（灵长类动物）（马穆） -B*17）11 （马木-A*01）12 （马穆-A*02）13，鼠标 （罗登蒂亚） （H-2Kd）14，015，狗 （卡尼沃拉） （DLA-88*50801）16，牛 （阿蒂奥达蒂拉） （博拉-A11）17和马 （佩里索德） （Eqca-N*00602 和 Eqca-N*00601）18可与异质β2米（表1）相结合。这些混合分子经常用于结构和功能研究。然而，杂交MHC I与异质β 2m的功能和结构研究方法尚未总结。同时，不同税种之间2米β互换的结构基础仍不明朗。
在此，总结了MHC I表达、重新折叠、结晶、晶体数据收集和结构测定的过程。此外，通过比较由同源和异质β 2米稳定下来的MHC I的结构构象，分析不同物种β 2米的潜在替代。这些方法将有助于进一步的MHC I结构研究和CD8+T 细胞免疫反应评估癌症和传染病。

<table>
	<tbody>
		<tr>
			<td>10 kDa MMCO membrane</td>
			<td>Merck millipore</td>
			<td>PLGC07610</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide</td>
			<td>LABLEAD</td>
			<td>A3291-500ml*5</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#215;Protein SDS Loading</td>
			<td>Novoprotein</td>
			<td>PM099-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>AMICON ULTRA-15 15ML-10 KDa cutoff</td>
			<td>Merck millipore</td>
			<td>UFC901096</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Inalco</td>
			<td>1758-9314</td>
			<td></td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Sigma</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3) strain</td>
			<td>TIANGEN</td>
			<td>CB105-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>MP</td>
			<td>219605580</td>
			<td>Wear suitable gloves and eye/face protection. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Solarbio</td>
			<td>D1070</td>
			<td>Gloves and goggles should be worn and operated in a ventilated kitchen. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.</td>
		</tr>
		<tr>
			<td>EDTA-2Na</td>
			<td>KeyGEN BioTECH</td>
			<td>KGT515500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin</td>
			<td>HUSHI</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>GSH</td>
			<td>Amresco</td>
			<td>0399-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>GSSG</td>
			<td>Amresco</td>
			<td>0524-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Amresco</td>
			<td>E424-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>h&#946;2m</td>
			<td>our lab</td>
			<td></td>
			<td>Zhang, S. et al. Structural basis of cross-allele presentation by HLA-A*0301 and HLA-A*1101 revealed by two HIV-derived peptide complexes. Mol Immunol. 49 (1-2), 395-401, (2011).</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Inalco</td>
			<td>1758-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine Hydrochloride</td>
			<td>Amresco</td>
			<td>0877-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Solarbio</td>
			<td>S8210</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Marker</td>
			<td>Fermentas</td>
			<td>26614</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Boao Rui Jing</td>
			<td>A112130</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex Increase 200 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Thermo</td>
			<td>17919</td>
			<td>Gloves and goggles should be worn and operated in a ventilated kitchen.</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Amresco</td>
			<td>0497-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Bioruler</td>
			<td>RH30056-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

stability and structure of bat major histocompatibility complex class i with heterologous &beta;2-microglobulin

主要的组织相容性复合物（MHC）在抗原肽的表达和T细胞对传染病和肿瘤发展的免疫反应中起着关键作用。杂交MHC I与异种β 2-微球蛋白（β 2米）从不同物种的替代可以稳定体外。这是研究哺乳动物MHC I的可行方法，当同源β 2米不可用时。同时，指出哺乳动物β 2米替代不会显著影响肽的表达。然而，对于与异种β 2-微球蛋白（β 2米）复合的混合MHC I的方法和技术的总结有限。在此，提出了在MHC I研究中评估异质β 2米替代的可行性的方法。这些方法包括准备表达结构：净化包容机构和重新折叠 MHC 综合体;确定蛋白质热稳定性;晶体筛选和结构确定。本研究为了解MHC I的功能和结构提供了建议，对传染病和肿瘤免疫治疗期间的T细胞反应评价也具有重要意义。

先前的工作报告说，HeV衍生的 HeV1 （DFANTFLP） 肽是由 Ptal-N*01：0110，19.在这里，评估了这种肽与Ptal-N*01：01的结合能力，同源蝙蝠β 2米（b+2...

Watch this Scientific Journal Video about Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous ÃŽÂ²2-Microglobulin at JoVE.com

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous ÃƒÅ½Ã‚Â²2-Microglobulin

该协议描述了通过从不同物种获得2-微球蛋白（β 2米）替代的潜在β获得稳定的主要组织相容性复合物（MHC）I类的实验方法。调查了由同源和异质β 2米稳定下来的MHC I的结构比较。

蝙蝠主要组织兼容性复杂类 I 的稳定性和结构与异质β2- 微球蛋白

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and ...

在本研究中，我们使用Bat MHC I类作为模型来总结方法，并评估与异质β-2微球蛋白复合的混合MHC I类的可行性。先前的研究表明，哺乳动物的B2M替代不会显著影响肽的表达。事实上，混合MHC类I可以激活一个类似的结构和功能，原来的分子。这些技术可用于促进MHC I在传染病和肿瘤免疫治疗期间组织反应评估的功能和结构化研究。对于大肠杆菌培养转化添加10纳米的质粒含有蝙蝠或人类MHC类Iβ两个微球蛋白氢链到100微升均衡悬浮和沐浴在冰中30分钟。在孵化结束时，热在42摄氏度下将细菌冲击90秒，并将培养回冰浴两分钟。接下来，在培养物中加入 800 微升的裂解肉汤，并在 37 摄氏度的摇动平台上摇动悬架 20 分钟，每分钟旋转 200 次。在孵化结束时，将100微升细菌传播到适当的抗生素耐药培养刀片上。第二天早上，挑选一个最近转化的细菌克隆，用三毫升LB用抗生素介质给克隆人接种疫苗。将文化放置在 37 摄氏度的摇动平台上 12 到 16 小时。第二天早上，用抗生素介质将500微升活性细菌转化为50毫升LB，并在一夜之间将细菌送回摇动孵化器。将剩余的活性细菌储存分成500微升的点名，并将每个点名添加到500微升的40%甘油中，储存温度为零下80摄氏度。为了产生大量的重组蛋白，第二天早上将细菌悬浮物以1比100的比例转化为两升LB，并将培养物返回到摇动孵化器，直到达到600纳米时0.6的吸收。然后，在培养中加入一毫摩尔IPTG，以诱导跨基因的表达。经过四到六个小时的诱导，将细菌培养转移到瓶子中进行离心，并将细菌细胞颗粒重新悬浮在每瓶60毫升PBS中。然后通过超声波细胞干扰器释放表达重组蛋白99次，每次声波6秒，间隔12秒，每次300瓦。为了内含体净化，通过离心收集声波细菌，并将颗粒重新悬浮在适当数量的洗涤缓冲液中，以便再进行两次离心。第二次清洗后，重新悬挂内含物并重新悬浮缓冲，并留出 20 微升的样本名言用于 SDS 页面，以测试内含物的纯度。将剩余的样品离心，并称重含有颗粒的内含物。将溶解缓冲器添加到颗粒中，最终浓度为每毫升缓冲液 30 毫克的内含体。使用磁性搅拌器在摄氏四度处缓慢搅拌溶液，直到内含物溶解在溶解缓冲区。丢弃沉淀物后，将内含物存放在零下20或零下80摄氏度。对于 MHC 复合重新折叠，在 250 到 300 毫升时添加 5 毫摩尔减少谷胱甘肽和 0.5 毫摩尔氧化谷胱甘肽，4 摄氏度重新折叠缓冲。在4摄氏度的磁搅拌器上慢慢搅拌溶液10至20分钟。对于 MHC 氢链和 β 两个微球蛋白稀释，将内含体装入一毫升注射器中，并将整个一毫升体积的内含体溶液注入搅拌棒附近的一升重新折叠缓冲器中，以获得快速高效的稀释。接下来，将每毫升5毫克肽溶解在二甲基硫化物中，并迅速将200微升肽注入刚刚证明的重新折叠溶液中。经过10至20分钟的缓慢搅拌后，将3毫升H链内含体注入新的一升重折叠缓冲区，让重新折叠在4摄氏度下进行8至10小时。要确定重新折叠的蛋白质浓度，在加压室中加入10千达顿多铜氧化酶膜的交换缓冲，并将缓冲区浓缩到30至50毫升体积。将重新折叠溶液转移到离心管中，并通过离心去除沉淀物。小心地将超生体转移到腔室中，并进一步将缓冲器浓缩到大约一毫升的最终体积。通过离心去除任何最终污染物，并将超生物转移到无菌管中。使用一个一千三百GL大小的排除列来净化蛋白质，在高峰期收集样本，并使用SDS页面分析它们。收集 MHC 复合峰值，将蛋白质浓缩到每毫升 15 毫克的最终浓度。然后稀释复合物至每毫升浓度7.5毫克。要使用坐滴蒸汽扩散技术对复杂的 MHC 和肽进行结晶，请将样品的 0.8 微升添加到商用晶体板中并密封板。将样品溶液与 100 微升的储层溶液相平衡，温度为 4 或 18 摄氏度，并使用显微镜在未来六个月内定期观察晶体生长情况。为了保护低温，在实验结束时，将晶体转移到含有20%甘油的储存溶液中，然后在100度开尔文气态氮气流中快速冷却样品。然后根据标准协议收集 X 射线衍射数据。要确定晶体复合物的结构，请打开香港 L 2000 软件包内 Denzo 程序中的高分辨率结构 X 射线衍射数据，并在蛋白质数据库中选择初始模型。要确定蛋白质的结构，请打开 CCP4 中相控器 MR 计划中的数据，并使用精制 X 射线模型进行初始联合细化。然后单独使用凤凰精炼和关节模式进行X射线单独细化，并采用联合中子进行X射线精炼。每轮改进后，手动检查模型对 Fo-Fc 和 2Fo-Fc 正核密度图在库特。在这些具有代表性的实验中，评估了亨德拉病毒衍生出亨德拉病毒一号肽的结合能力，即用同源蝙蝠β两种微球蛋白和异种人类β两种微球蛋白击打MHC I类氢链。在这两种分析中，高分辨率晶体都是通过测试版两个微球蛋白光链的重新自然形成的。在没有β两个微球蛋白的情况下，这些复合物不会形成。在MHCI类氢链亨德拉病毒一、人类β二类微球蛋白结构中， 人类β两种微球蛋白的保存残留物H31、D53、W60和Y63，对应于蝙蝠β两种微球蛋白，与肽结合槽底部接触，保存Q8、Y10、R12和24残留物，对应于与α三域结合的β两个微球蛋白。在整体蝙蝠和人类结构中，氢链中1至184个残留物的平均根系均为0.248，表明这两个复合物之间没有区别。肽对齐的结构表明，亨德拉病毒在这两个复合物中的一个肽的确认是相当相似的。序列对齐还表明，来自不同物种的β两种微球蛋白的氨基酸受到高度保护。重要的是要以高报告效率为对手提供综合体。因此，选择合适的β两个微球蛋白和使用高度纯化的包容体至关重要。该协议可用于通过其他物种中潜在的β两种微球蛋白替代获得稳定的MTC I复合物，并存在它们是MTC I的结构和功能。从这些研究中获得的数据可用于评估传染病和肿瘤免疫治疗期间的T细胞反应。

Research

JoVE Journal

Immunology and Infection

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &beta;2-Microglobulin

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

纯净小鼠的B细胞的诱导和评估类开关重组

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

科研

免疫与感染

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Real-time Live Imaging of T-cell Signaling Complex Formation

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

在小鼠小腿上应用一致的按摩样扰动，并监测由此产生的肌肉压力变化

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

按流细胞测量评估主要穆林河马神经元的主要组织相容性综合类 I 的表达

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A ...