小尾寒羊β2微球蛋白的表达及其二级结构预测分析

doi:10.16431/j.cnki.1671-7236.2020.07.003

摘要/Abstract

摘要： 为研究小尾寒羊绵羊白细胞抗原Ⅰ（Ovis aries leukocyte antigen classⅠ，OLAⅠ）轻链四聚体前体链β₂微球蛋白（β₂-microglobulin，β₂m）的结构和功能，根据GenBank中公布的绵羊β₂m基因设计特异性引物，提取小尾寒羊全血中的RNA并运用RT-PCR方法扩增绵羊β₂m基因，将扩增的绵羊β₂m基因克隆到pMD18-T载体，筛选出阳性克隆菌pMD18T-OLAⅠ-β₂m，经双酶切后与表达载体pET-28a（+）连接，再转化大肠杆菌BL21（DE3）感受态细胞中构建pET-28a（+）-OLAⅠ-β₂m重组表达菌，经IPTG诱导表达，SDS-PAGE检测目的蛋白大小及表达情况；运用SOMPA在线软件预测OLAⅠ-β₂m蛋白的二级结构。PCR扩增结果显示，目的基因大小为357 bp，与理论值相符，扩增片段成功克隆到pMD18-T载体，经EcoR Ⅰ和Hind Ⅲ双酶切筛选及测序，成功获得阳性克隆菌株pMD18-T-OLAⅠ-β₂m，插入的目的片段大小为357 bp。阳性克隆菌株与表达载体pET-28a（+）经EcoR Ⅰ和Hind Ⅲ双酶切后连接，转化大肠杆菌BL21（DE3）感受态细胞后获得pET-28a（+）-OLAⅠ-β₂m重组表达菌，经IPTG诱导表达，Western blotting检测目的蛋白大小为17.3 ku，目的蛋白在大肠杆菌中主要以包涵体的形式表达，经洗涤、变性、纯化、初步获得SDS-PAGE纯化的OLAⅠ-β₂m蛋白；PORTER在线软件预测OLAⅠ-β₂m蛋白的二级结构元件α-螺旋（Hh）、β-折叠（Ee）和无规则卷曲（Cc）分别占22.03%、22.03%和55.93%。本研究成功构建了小尾寒羊β₂m基因的pET-28a（+）重组表达体系，运用SOMPA在线软件预测OLAⅠ-β₂m的二级结构，为下一步绵羊OLAⅠ类分子四聚体的构建奠定基础。

关键词: β₂微球蛋白; 小尾寒羊; 主要组织相容性复合体; 包涵体

Abstract: In order to study the structure and function of Small-tailed Han sheep leukocyte antigen (OLA) class Ⅰ tetramer light chain β₂-microglobulin (β₂m).A pair of specific primers based on the published sequence of this gene was designed and the cDNA of full coding region for β₂m precursor was obtained by RT-PCR from the Small-tailed Han sheep whole blood.The Small-tailed Han sheep β₂m gene was cloned into the pMD18-T vector and the positive clones of pMD18T-OLAⅠ-β₂m were selected.After double digestion,the β₂m gene in positive clones was further ligated into the pET-28a(+) expression vector and transformed into E.coli BL21(DE3) to construct the recombinant strain of pET-28a(+)-OLAⅠ-β₂m.Then the targeted protein was detected by SDS-PAGE within induction of IPTG.The secondary structure of the OLAⅠ-β₂m protein was predicted and analyzed by SOMPA software.The PCR results showed that the length of OLAⅠ-β₂m was about 357 bp which was consistent with the theoretical value.The amplified fragment was successfully cloned into pMD18-T vector,and the positive clones were verified by EcoR Ⅰ and Hind Ⅲ digestion and DNA sequencing analysis.The cut β₂m gene from pMD18T-OLAⅠ-β₂m was inserted into pET-28a(+) and transformed into E.coli BL21(DE3) successfully.After IPTG induction,the expressed protein was detected by western blotting and SDS-PAGE,the results showed that the target protein (17.3 ku) was expressed efficiently with inclusion body.Finally,the secondary structure α-helix,extended strand and random coil of the target protein was 22.03%,22.03% and 55.93%,respectively.In this study,the recombinant tetramer precursor of OLAⅠ-β₂m light chain was constructed into pET-28a(+) expression line successfully,and the secondary structure of the OLAⅠ-β₂m protein was predicted and analyzed by SOMPA software,which would lay a solid foundation for constructing the OLAⅠtetramer of Small-tailed Han sheep.

Key words: β₂-microglobulin; Small-tailed Han sheep; major histocompatibility complex; inclusion body

中图分类号:

S852.62

王战红, 赵志荀, 吴国华, 朱学亮, 黄彩云, 祁斌亮, 赵芳燕, 张志东, 张强. 小尾寒羊β₂微球蛋白的表达及其二级结构预测分析[J]. 中国畜牧兽医, 2020, 47(7): 1990-1996.

WANG Zhanhong, ZHAO Zhixun, WU Guohua, ZHU Xueliang, HUANG Caiyun, QI Binliang, ZHAO Fangyan, ZHANG Zhidong, ZHANG Qiang. Expression and Secondary Structure Analysis of β₂-microglobulin in Small-tailed Han Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 1990-1996.

参考文献

[1] ALTMAN J D,MOSS P A,GOULDER P J,et al.Phenotypic analysis of antigen-specific T lymphocytes[J].Science,1996,274(5284):94-96.
[2] APPAY V,ROWLAND-JONES S L.The assessment of antigen-specific CD8⁺ T cells through the combination of MHC class Ⅰ tetramer and intracellular staining[J].Journal of Immunological Methods,2002,268(1):9-19.
[3] HUGUES S,MALHERBE L,FILIPPI C,et al.Generation and use of alternative multimers of peptide/MHC complexes[J].Journal of Immunological Methods,2002,268(1):83-92.
[4] 郑世军.动物分子免疫学[M].北京:中国农业出版社,2015. ZHENG S J.Veterinary Molecular Immunology[M].Beijing:China Agriculture Press,2015.(in Chinese)
[5] PRASANNA S J,NANDI D.The MHC-encoded class Ⅰ molecule,H-2Kk,demonstrates distinct requirements of assembly factors for cell surface expression:Roles of TAP,Tapasin and beta2-microglobulin[J].Molecular Immunology,2004,41(10):1029-1045.
[6] MACHOLD R P,ANDREE S,VAN KAER L,et al.Peptide influences the folding and intracellular transport of free major histocompatibility complex class Ⅰ heavy chains[J].Journal of Experimental Medicine,1995,181(3):1111-1122.
[7] GARBOCZI D N,HUNG D T,WILEY D C.HLA-A2-peptide complexes:Refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides[J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(8):3429-3433.
[8] CHEN L M,VERITY N J,CHAI K X.Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)[J].BMC Cancer,2009,22(9):377.
[9] NOMURA T,HUANG W C,ZHAU H E,et al.Beta2-microglobulin promotes the growth of human renal cell carcinoma through the activation of the protein kinase A,cyclic AMP-responsive element-binding protein,and vascular endothelial growth factor axis[J].Clinical Cancer Research,2006,12(24):7294-7305.
[10] LI L,DONG M,WANG X G.The implication and significance of beta 2 microglobulin:A conservative multifunctional regulator[J].Chinese Medical Journal (English),2016,129(4):448-455.
[11] 朴文花,何豫,席宏丽,等.HLA-A2肽四聚体的构建及其在乙、丙型肝炎中的初步应用[J].中华医学杂志,2004,21:62-66. PIAO W H,HE Y,XI H L,et al.Construction of HLA-A2-peptide tetramer and application in HBV/HCV infection[J].National Medical Journal of China,2004,21:62-66.(in Chinese)
[12] VINCENT B G,YOUNG E F,BUNTZMAN A S,et al.Toxin-coupled MHC class Ⅰ tetramers can specifically ablate autoreactive CD8⁺T cells and delay diabetes in nonobese diabetic mice[J].Journal of Immunology,2010,184(8):4196-204.
[13] BHARAT A,BENSHOFF N,FLEMING T P,et al.Characterization of the role of CD8⁺T cells in breast cancer immunity following mammaglobin-A DNA vaccination using HLA-class-Ⅰ tetramers[J].Breast Cancer Research and Treatment,2008,110(3):453-463.
[14] 刘光亮.BF2/肽四聚体的构建及其在传染性支气管炎病毒特异性T细胞反应评价中的应用[D].北京:中国农业科学院,2007. LIU G L.Construction of BF2/peptide tetramer and assessment of the infectious bronchitis virus-specific T cells by using the prepared BF2/peptide tetramer[D].Beijing:Chinese Academy of Agricultural Sciences,2007.(in Chinese)
[15] 李子彬.荷包猪SLA-2/肽四聚体构建与功能检测[D].长春:吉林农业大学,2015. LI Z B.Preparation and functional detection of Hebao pig SLA-2/peptide tetramer[D].Changchun:Jilin Agricultural University,2015.(in Chinese)
[16] KOZLOWSKI S,TAKESHITA T,BOEHNCKE W H,et al.Excess beta 2 microglobulin promoting functional peptide association with purified soluble class Ⅰ MHC molecules[J].Nature,1991,349(6304):74-77.
[17] OTTEN G R,BIKOFF E,RIBAUDO R K,et al.Peptide and beta 2-microglobulin regulation of cell surface MHC class Ⅰ conformation and expression[J].The Journal of Immunology,1992,148(12):3723-3732.
[18] SHIELDS M J,ASSEFO N,HODGSON W,et al.Characterization of the interactions between MHC class Ⅰ subunits:A systematic approach for the engineering of higher affinity variants of beta 2-microglobulin[J].The Journal of Immunology,1998,160(5):2297-2307.
[19] SHIELDS M J,MOFFAT L E,RIBAUDO R K.Functional comparison of bovine,murine,and human beta2-microglobulin:Interactions with murine MHC Ⅰ molecules[J].Molecular Immunology,1998,35(14):919-928.
[20] 何贤辉,徐丽慧,刘毅,等.人β₂-微球蛋白基因克隆及其在大肠杆菌中的高效表达[J].生物工程学报,2004,1:99-103. HE X H,XU L H,LIU Y,et al.Cloning of human β₂-microglobulin gene and its high expression in Escherichia coli[J].Chinese Journal of Biotechnology,2004,1:99-103.(in Chinese)
[21] 刘光亮,童铁钢,孟庆文,等.鸡β₂微球蛋白基因克隆及其在大肠杆菌中的高效表达与纯化[J].中国兽医学报,2007,5:649-652. LIU G L,TONG T G,MENG Q W,et al.Cloning,expression and purification of chicken β₂-microglobulin in Escherichia coli[J].Chinese Journal of Veterinary Science,2007,5:649-652.(in Chinese)
[22] 高凤山,夏春,张强,等.大肠杆菌表达的重组猪β₂微球蛋白二级结构的圆二色谱分析[J].微生物学报,2009,49(12):1596-1600. GAO F S,XIA C,ZHANG Q,et al.Analyzing secondary structure of β₂ microglobulin in Escherichia coli with circular dichroism spectrum[J].Acta Microbiologica Sinica,2009,49(12):1596-1600.(in Chinese)
[23] KOHNO T,CARMICHAEL D F,SOMMER A,et al.Refolding of recombinant proteins[J].Methods in Enzymol,1990,185(2):187-195.

小尾寒羊β₂微球蛋白的表达及其二级结构预测分析

Expression and Secondary Structure Analysis of β₂-microglobulin in Small-tailed Han Sheep

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