JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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면역학 및 감염병학

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

Published: March 10, 2021
doi:
10.3791/61462
, , , , , , , , , ,
1School of Laboratory Medicine and Life Sciences,Wenzhou Medical University, 2NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention, 3Faculty of Health Sciences,University of Macau, 4CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology,Chinese Academy of Sciences, 5Savaid Medical School,University of Chinese Academy of Sciences

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 表达结构的准备 从NCBI数据库中检索MHC I类基因（包括预测基因）的序列。 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。 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。 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?…

Representative Results

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…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

10 kDa MMCO membrane Merck millipore PLGC07610
30% Acrylamide LABLEAD A3291-500ml*5
5×Protein SDS Loading Novoprotein PM099-01A
AMICON ULTRA-15 15ML-10 KDa cutoff Merck millipore UFC901096
Ampicillin Inalco 1758-9314
APS Sigma A3678-100G
BL21(DE3) strain TIANGEN CB105-02
DMSO MP 219605580 Wear suitable gloves and eye/face protection. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
DTT Solarbio D1070 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.
EDTA-2Na KeyGEN BioTECH KGT515500
Glycerin HUSHI 10010618
GSH Amresco 0399-250G
GSSG Amresco 0524-100G
Guanidine hydrochloride Amresco E424-5KG
hβ2m our lab 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).
IPTG Inalco 1758-1400
L-Arginine Hydrochloride Amresco 0877-5KG
NaCl Solarbio S8210
Protein Marker Fermentas 26614
SDS Boao Rui Jing A112130
Superdex Increase 200 10/300 GL GE Healthcare 28990944
TEMED Thermo 17919 Gloves and goggles should be worn and operated in a ventilated kitchen.
Tris-HCl Amresco 0497-5KG
Triton X-100 Bioruler RH30056-100mL
Tryptone Oxoid LP0042
Yeast extract Oxoid LP0021
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Tags

Keywords: Bat MHC Class IHeterologous β2-microglobulinHybrid MHC Class IE. Coli TransformationRecombinant Protein ExpressionIPTG InductionBacterial Cell PelletPBS
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Zhang, D., Liu, K., Lu, D., Wang, P., Zhang, Q., Liu, P., Zhao, Y., Chai, Y., Lyu, J., Qi, J., Liu, W. J. Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous β2-Microglobulin. J. Vis. Exp. (169), e61462, doi:10.3791/61462 (2021).
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