牛早幼粒细胞白血病蛋白的原核表达及单克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.09.029

摘要/Abstract

摘要： 【目的】牛早幼粒细胞白血病蛋白(promyelocytic leukemia protein，PML)是细胞核亚结构PML核体(nuclear bodies，NBs)的骨架蛋白，在抗病毒内源性免疫中发挥重要作用。本研究旨在制备牛PML单克隆抗体，为研究PML NBs结构和功能准备物质材料。【方法】构建表达bPML基因截短体的重组质粒pET-32a-bPML，将重组质粒转化大肠杆菌Transetta(DE3)感受态细胞，IPTG诱导表达，利用SDS-PAGE和Western blotting对表达产物进行分析。将经镍柱亲和层析纯化后的重组蛋白免疫小鼠，制备单克隆抗体，应用ELISA方法鉴定单克隆抗体类/亚类和型，进一步鉴定该单克隆抗体识别的抗原表位；通过检测不同种属来源的细胞检测单克隆抗体的特异性；利用该单克隆抗体对外源转染牛PML的细胞进行Western blotting和间接免疫荧光试验(IFA)检测；利用该单克隆抗体建立的IFA检测不同的牛源细胞内源PML定位情况。【结果】牛PML在大肠杆菌中可溶性表达，分子质量大小为50 ku；以此纯化蛋白为免疫原制备出1株抗牛PML单克隆抗体bPML-2G5，其为IgG1亚类，轻链为Kappa型，识别表位位于牛PML结构域⁷⁸EQPRPSTSRA⁸⁸；Western blotting和IFA分析结果显示，bPML-2G5不仅可特异性识别外源转染的牛PML，而且还能特异性识别牛肾细胞、牛鼻甲骨细胞、牛胚气管细胞的胞核内核体结构中的PML。【结论】本研究成功制备了1株分泌牛PML单克隆抗体的杂交瘤细胞株，其分泌的单克隆抗体可用于检测外源性及内源性表达的牛PML。

关键词: 牛早幼粒细胞白血病蛋白; 原核表达; 蛋白纯化; 单克隆抗体

Abstract: 【Objective】 Bovine promyelocytic leukemia protein (PML)，a constituent of nuclear substructure called PML nuclear bodies (PML NBs), was crucial for antiviral intrinsic immunity.In this study, monoclonal antibody against bovine PML was generated to facilitate further investigation into the structure and function of PML NBs. 【Method】 Recombinant plasmid pET-32a-bPML expressing bPML gene truncator was constructed.The recombinant plasmid was transformed into Escherichia coli Transetta (DE3) competent cells, and induced by IPTG, and the expression products were analyzed by SDS-PAGE and Western blotting.The recombinant protein purified by nickel column affinity chromatography was utilized for immunization of mice, and monoclonal antibody was prepared.ELISA was applied to determine the classes/subclasses and subtypes of the monoclonal antibody, and the epitope recognized by the monoclonal antibody was further identified.The specificity of monoclonal antibody was detected by detecting cells of different species origin.The monoclonal antibody against bovine PML was analyzed by Western blotting and indirect immunofluorescence assay (IFA) to detect of exogenously transfected bovine PML cells.IFA established by this monoclonal antibody was used to detect the endogenous PML localization in different bovine-derived cells. 【Result】 Bovine PML was solubly expressed in Escherichia coli with a molecular weight size of 50 ku, a monoclonal antibody targeting bovine PML, bPML-2G5, was prepared from this purified protein as an immunogen.bPML-2G5 belonged to the IgG1 subclass, its light chain was Kappa type.The epitope recognized by bPML-2G5 was located in the structural domain ⁷⁸EQPRPSTSRA⁸⁸ of the bovine PML.Western blotting and IFA analysis demostrated that bPML-2G5 not only specifically recognized the exogenously transfected bovine PML, but also specifically recognized PML localized within PML NBs in bovine kidney cells, bovine nasal turbinate cells, and bovine embryonic tracheal cells. 【Conclusion】 In this study, a hybridoma cell line secreting bovine PML monoclonal antibody was successfully prepared, and its secreted monoclonal antibody could be used to detect exogenously and endogenously expressed bovine PML proteins.

Key words: bovine promyelocytic leukemia protein; prokaryotic expression; protein purification; monoclonal antibody

中图分类号:

S852.4

程晶, 陈沛霖, 郭禹, 崔锦蔷, 江波, 周林宜, 刘文晓, 李焕荣, 李永清. 牛早幼粒细胞白血病蛋白的原核表达及单克隆抗体制备[J]. 中国畜牧兽医, 2024, 51(9): 4014-4024.

CHENG Jing, CHEN Peilin, GUO Yu, CUI Jinqiang, JIANG Bo, ZHOU Linyi, LIU Wenxiao, LI Huanrong, LI Yongqing. Prokaryotic Expression and Monoclonal Antibody Preparation of Bovine Promyelocytic Leukemia Protein[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 4014-4024.

参考文献

[1] UGGE M,SIMONI M,FRACASSI C,et al.PML isoforms:A molecular basis for PML pleiotropic functions[J].Trends in Biochemical Sciences,2022,47(7):609-619.
[2] SHIM M S,NETTESHEIM A,HIRT J,et al.The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress[J].Autophagy,2020,16(7):1248-1261.
[3] SANYAL A,WALLASCHEK N,GLASS M,et al.The ND10 complex represses lytic Human herpesvirus 6A replication and promotes silencing of the viral genome[J].Viruses,2018,10(8)：803-815.
[4] WAGENKNECHT N,REUTER N,SCHERER M,et al.Contribution of the major ND10 proteins PML,hDaxx and Sp100 to the regulation of Human cytomegalovirus latency and lytic replication in the monocytic cell line THP-1[J].Viruses,2015,7(6):2884-2907.
[5] DUTRIEUX J,MAARIFI G,PORTILHO D M,et al.PML/TRIM19-dependent inhibition of retroviral reverse-transcription by Daxx[J].PLoS Pathogens,2015,11(11):e1005280.
[6] ALANDIJANY T,ROBERTS A P E,CONN K L,et al.Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection[J].PLoS Pathogens,2018,14(1):e1006769.
[7] MA Y,LI J,DONG H,et al.PML body component Sp100A restricts wild-type Herpes simplex virus 1 infection[J].Journal of Virology,2022,96(8):279-293.
[8] RODRÍGUEZ M C,DYBAS J M,HUGHES J,et al.The HSV-1 ubiquitin ligase ICP0:Modifying the cellular proteome to promote infection[J].Virus Research,2020,28(5):198-215.
[9] SEELER J S,DEJEAN A.SUMO and the robustness of cancer[J].Nature Reviews Cancer,2017,17(3):184-197.
[10] BERNARDI R,PANDOLFI P P.Structure,dynamics and functions of promyelocytic leukaemia nuclear bodies[J].Nature Reviews Molecular Cell Biology,2007,8(12):1006-1016.
[11] NISOLE S,MAROUI M A,MASCLE X H,et al.Differential roles of PML isoforms[J].Frontiers in Oncology,2013,3(11):125-139.
[12] CHEN Y,WRIGHT J,MENG X,et al.Promyelocytic leukemia protein isoform Ⅱ promotes transcription factor recruitment to activate interferon beta and interferon-responsive gene expression[J].Molecular and Cellular Biology,2015,35(10):1660-1672.
[13] EL ASMI F,MAROUI M A,DUTRIEUX J,et al.Implication of PMLIV in both intrinsic and innate immunity[J].PLoS Pathogens,2014,10(2):e1003975.
[14] LI C,PENG Q,WAN X,et al.C-terminal motifs in promyelocytic leukemia protein isoforms critically regulate PML nuclear body formation[J].Journal of Cell Science,2017,130(20):3496-3506.
[15] YU C,XU A,LANG Y,et al.Swine promyelocytic leukemia isoform Ⅱ inhibits Pseudorabies virus infection by suppressing viral gene transcription in promyelocytic leukemia nuclear bodies[J].Journal of Virology,2020,119(7)：107-120.
[16] BIENIASZ P D.Intrinsic immunity:A front-line defense against viral attack[J]. Nature Immunology,2004,5(11):1109-1115.
[17] FULL F,ENSSER A.Early nuclear events after herpesviral infection[J].Journal of Clinical Medicine,2019,8(9):1408.
[18] LALLEMANDB V,DE THÉ H.PML nuclear bodies:From architecture to function[J].Current Opinion in Cell Biology,2018,52(5):154-161.
[19] REICHELT M,WANG L,SOMMER M,et al.Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against Varicella-zoster virus[J].PLoS Pathogens,2011,7(2):126-135.
[20] STEPP W H,MEYERS J M,MCBRIDE A A.Sp100 provides intrinsic immunity against Human papillomavirus infection[J].Applied and Environmental Microbiology,2013,4(6):813-835.
[21] MITCHELL A M,HIRSCH M L,LI C,et al.Promyelocytic leukemia protein is a cell-intrinsic factor inhibiting Parvovirus DNA replication[J].Journal of Virology,2014,88(2):925-936.
[22] KUROKI M,ARIUMI Y,HIJIKATA M,et al.PML tumor suppressor protein is required for HCV production[J].Biochemical and Biophysical Research Communications,2013,430(2):592-597.
[23] 许傲天.猪PML的抗病毒作用及伪狂犬病毒对其产生的拮抗机制研究[D].北京：中国农业大学,2015. XU A T.Study on antiviral effects of swine promyelocytic lekumia protein (PML) and the mechanism that Pseudorabies virus escaped swine PML antiviral effects[D].Beijing:China Agricultural University,2015.(in Chinese)
[24] 王文婕,茹鹏辉,郁川,等.猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J].中国畜牧兽医,2023,50(4):1511-1521. WANG W J，RU P H,YU C,et al.Prokaryotic expression of VP2 protein of Luoyang Feline panleukopenia virus isolate and polyclonal antibody preparation[J].China Animal Husbandry & Veterinary Medicine, 2023,50(4):1511-1521.(in Chinese)
[25] 罗世美,赵瑶,马鲜平,等.蓝舌病病毒NS4蛋白原核表达及多克隆抗体制备[J].中国畜牧兽医,2023,50(7):2923-2930. LUO S M，ZHAO Y,MA X P, et al.Prokaryotic expression and polyclonal antibody preparation of Bluetongue virus NS4 protein[J].China Animal Husbandry & Veterinary Medicine, 2023,50(7):2923-2930.(in Chinese)