鸡CTLA-4蛋白在昆虫细胞中的表达及其单克隆抗体的制备

doi:10.11843/j.issn.0366-6964.2023.06.036

Abstract

Abstract: The aim of this study was to prepare monoclonal antibody specific to chicken CTLA-4, and provide useful tools for research on the poultry immune checkpoint and vaccine development. Baculovirus expression system was used to express chicken CTLA-4 protein in vitro. The monoclonal antibodies against chicken CTLA-4 protein were prepared by fusion between SP2/0 cells and spleen cells from the 8-week-old Balb/c mice immunized with the expressed chicken CTLA-4 protein. The charcterization of monoclonal antibodies were analyzed by indirect immunofluorescence, Western blot and flow cytometry. The recombinant baculovirus expressing CTLA-4 protein in Sf9 insect cells was constructed. Three monoclonal antibodies against chicken CTLA-4 protein were successfully obtained, named as mAb-CTLA4-3D7, mAb-CTLA4-5A4 and mAb-CTLA4-6A12. The subclass of mAb-CTLA4-3D7 was IgG2a and type Lambda; The subclass of mAb-CTLA4-5A4 was IgG3 and type Kappa; The subclass of mAb-CTLA4-6A12 was IgG2a and type Kappa. All three monoclonal antibodies could react with DF-1 cells transfected with pCAGGS-CTLA-4-Flag and Sf9 insect cells infected with rBac-CTLA-4. Among them, mAb-CTLA4-3D7 had a good binding activity with chicken PBMC and could react to CD3⁺T lymphocytes, with the binding rate of about 16%. In this study, monoclonal antibodies against chicken CTLA-4 protein were successfully developed, and their reactivity and biological characteristics were analyzed, which provided a reference for immune checkpoints in the pathogenesis, signaling pathways, vaccine development and other research fields of avian diseases.

Key words: chicken CTLA-4, baculovirus, monoclonal antibody, biological characteristics

CLC Number:

S852.4

LIU Qing, WU Shaopeng, SHI Bin, SHAO Hongxia, QIAN Kun, YE Jianqiang, QIN Aijian. Expression of Chicken CTLA-4 Protein in Insect Cells and the Preparation of Its Monoclonal Antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2596-2604.

References 31

[1]	LINSLEY P S,GREENE J L,TAN P,et al.Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes[J].J Exp Med,1992,176(6):1595-1604.
[2]	GOUGH S C L,WALKER L S K,SANSOM D M.CTLA4 gene polymorphism and autoimmunity[J].Immunol Rev,2005,204(1):102-115.
[3]	LINSLEY P S,BRADY W,URNES M,et al.CTLA-4 is a second receptor for the B cell activation antigen B7[J].J Exp Med,1991,174(3):561-569.
[4]	ZHANG Y P,ZHENG J K.Functions of immune checkpoint molecules beyond immune evasion[M]//XU J.Regulation of Cancer Immune Checkpoints.Singapore:Springer,2020:201-226.
[5]	MITSUIKI N,SCHWAB C,GRIMBACHER B.What did we learn from CTLA-4 insufficiency on the human immune system?[J].Immunol Rev,2019,287(1):33-49.
[6]	ZHANG A L,REN Z H,TSENG K F,et al.Dual targeting of CTLA-4 and CD47 on Treg cells promotes immunity against solid tumors[J].Sci Transl Med,2021,13(605):eabg8693.
[7]	ROWSHANRAVAN B,HALLIDAY N,SANSOM D M.CTLA-4:a moving target in immunotherapy[J].Blood,2018,131(1):58-67.
[8]	WALUNAS T L,LENSCHOW D J,BAKKER C Y,et al.CTLA-4 can function as a negative regulator of T cell activation[J].Immunity,1994,1(5):405-413.
[9]	PARVIZI P,ANDRZEJEWSKI K,READ L R,et al.Expression profiling of genes associated with regulatory functions of T-cell subsets in Marek's disease virus-infected chickens[J].Avian Pathol,2010,39(5):367-373.
[10]	JARAHIAN M,WATZL C,FOURNIER P,et al.Activation of natural killer cells by newcastle disease virus hemagglutinin-neuraminidase[J].J Virol,2009,83(16):8108-8121.
[11]	YU H L,ZOU W B,MI C H,et al.Research Note:Expression of T cell-related cytokines in chicken cecal and spleen tissues following Eimeria tenella infection in vivo[J].Poult Sci,2021,100(7):101161.
[12]	RUAN Y,WANG Y,GUO Y P,et al.T cell subset profile and inflammatory cytokine properties in the gut-associated lymphoid tissues of chickens during infectious bursal disease virus (IBDV) infection[J].Arch Virol,2020,165(10):2249-2258.
[13]	MO X,ZHANG H H,PRESTON S,et al.Interferon-γ signaling in melanocytes and melanoma cells regulates expression of CTLA-4[J].Cancer Res,2018,78(2):436-450.
[14]	LEE E Q.Immune checkpoint inhibitors in GBM[J].J Neurooncol,2021,155(1):1-11.
[15]	WOLCHOK J D,NEYNS B,LINETTE G,et al.Ipilimumab monotherapy in patients with pretreated advanced melanoma:A randomised,double-blind,multicentre,phase 2,dose-ranging study[J].Lancet Oncol,2010,11(2):155-164.
[16]	TANG C L,YANG J F,PAN Q,et al.Anti-CTLA-4 monoclonal antibody improves efficacy of the glyceraldehyde-3-phosphate dehydrogenase protein vaccine against Schistosoma japonicum in mice[J].Parasitol Res,2019,118(7):2287-2293.
[17]	LIU L N,WANG Y H,MIAO L,et al.Combination immunotherapy of MUC1 mRNA Nano-vaccine and CTLA-4 blockade effectively inhibits growth of triple negative breast cancer[J].Mol Ther,2018,26(1):45-55.
[18]	IRONS S L,CHAMBERS A C,LISSINA O,et al.Protein production using the baculovirus expression system[J].Curr Protoc Protein Sci,2018,91:5.5.1-5.5.22.
[19]	SCHNEIDER H,SMITH X,LIU H B,et al.CTLA-4 disrupts ZAP70 microcluster formation with reduced T cell/APC dwell times and calcium mobilization[J].Eur J Immunol,2008,38(1):40-47.
[20]	周艳,董文.CTLA-4单克隆抗体——靶向被动免疫治疗肿瘤[J].临床肿瘤学杂志,2013,18(3):268-272.ZHOU Y,DONG W.CTLA-4 monoclonal antibody-targeted passive immunotherapy for tumor[J].Chinese Clinical Oncology,2013,18(3):268-272.(in Chinese)
[21]	CARLINO M S,LARKIN J,LONG G V.Immune checkpoint inhibitors in melanoma[J].Lancet,2021,398(10304):1002-1014.
[22]	OWONIKOKO T K,PARK K,GOVINDAN R,et al.Nivolumab and ipilimumab as maintenance therapy in extensive-disease small-cell lung cancer:CheckMate 451[J].J Clin Oncol,2021,39(12):1349-1359.
[23]	LEACH D R,KRUMMEL M F,ALLISON J P.Enhancement of antitumor immunity by CTLA-4 blockade[J].Science,1996,271(5256):1734-1736.
[24]	ZHENG P,WU Y,GUO Y,et al.B7-CTLA4 interaction enhances both production of antitumor cytotoxic T lymphocytes and resistance to tumor challenge[J].Proc Natl Acad Sci U S A,1998,95(11):6284-6289.
[25]	HURWITZ A A,FOSTER B A,KWON E D,et al.Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade[J].Cancer Res,2000,60(9):2444-2448.
[26]	YANG Y,DONG M L,HAO X L,et al.Revisiting cellular immune response to oncogenic Marek’s disease virus:the rising of avian T-cell immunity[J].Cell Mol Life Sci,2020,77(16):3103-3116.
[27]	唐春莲,王金松.CTLA-4单克隆抗体的研究进展[J].细胞与分子免疫学杂志,2013,29(7):769-771.TANG C L,WANG J S.Research progress of CTLA-4 monoclonal antibody[J].Chinese Journal of Cellular and Molecular Immunology,2013,29(7):769-771.(in Chinese)
[28]	ROTTE A.Combination of CTLA-4 and PD-1 blockers for treatment of cancer[J].J Exp Clin Cancer Res,2019,38(1):255.
[29]	CHAMBERS A C,AKSULAR M,GRAVES L P,et al.Overview of the baculovirus expression system[J].Curr Protoc Protein Sci,2018,91:5.4.1-5.4.6.
[30]	YAO Q X,FISCHER K P,MOTYKA B,et al.Identification of cytotoxic T-lymphocyte-associated antigen-4(CTLA-4) isoforms in the Pekin duck[J].Dev Comp Immunol,2010,34(7):749-758.
[31]	WANG C,HAO C F,DAI K,et al.Occupational low-dose radiation affects the expression of immune checkpoint of medical radiologists[J].Int J Environ Res Public Health,2022,19(12):7105.

Expression of Chicken CTLA-4 Protein in Insect Cells and the Preparation of Its Monoclonal Antibody

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 31

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LIU Chuanxia, WANG Xiao, LI Xuewen, BAO Miaofei, LI Tingting, CHEN Xin, WENG Changjiang, ZHENG Jun. Preparation of Monoclonal Antibody of African Swine Fever Virus pE120R [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 388-394.
[2]	LIU Taoxue, SU Bingqian, QI Yanli, GUO Jiangtao, LIU Zhonghu, CHU Beibei, WANG Jiang, ZENG Lei. Preparation of the Monoclonal Antibody against the African Swine Fever Virus p30 Protein and Identification of the Antigenic Epitope [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3415-3423.
[3]	YUAN Li, SUN Yangyang, ZHANG Lujie, ZHANG Jie, SUN Haifeng, BAI Juan, JIANG Ping. Production and Epitope Characterization of Monoclonal Antibodies against GP3 Protein of Porcine Reproductive and Respiratory Syndrome Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3424-3434.
[4]	LI Zhaoyan, GAO Jiang, GUO Shihui, ZHAO Ruqian, MA Wenqiang. Advances in Detection Methods and Control Solutions for Feline Allergens [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2272-2279.
[5]	CAO Liyan, KONG Xiangyu, LI Xiangtong, SUO Xuepeng, DUAN Yueyue, YUAN Cong, SHI Lei, ZHANG Yu, MA Guoxiang, ZHENG Haixue, WANG Qi. Preparation and Sequence Analysis of Monoclonal Antibody against the Nucleocapsid Protein of Porcine Acute Diarrhea Syndrome Coronavirus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2487-2497.
[6]	ZHAI Yunyi, YUAN Ye, LI Junmei, TIAN Lulu, DIAO Ziyang, LI Bin, CHEN Jialu, ZHOU Dong, JIN Yaping, WANG Aihua. Preparation and Preliminary Application of Monoclonal Antibody to Brucella Outer Membrane Protein 16 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2083-2091.
[7]	LI Xiaohan, YANG Fuchun, LIU Rui, GAO Li, CUI Hongyu, ZHANG Yanping, LIU Changjun, QI Xiaole, WANG Xiaomei, GAO Yulong, LI Kai. Construction of Recombinant Herpesvirus of Turkey Expressing Infectious Bursal Disease Virus VP2 Gene and Analysis of Its Biological Characteristics [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 656-662.
[8]	HUANG Jingjie, LI Miao, CHEN Yingxian, ZHONG Yalan, ZHANG Tingting, JIANG Tingchaonan, LI Jiancheng. Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Fluorquinolones based on Immunomagnetic Bead Purification [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 766-778.
[9]	ZHANG Jiaqi, JIA Xining, ZHOU Qun, SONG Xin, ZHU Chenxi, LI Gege, ZHANG Bin. Construction of BVDV-1 Virus-like Particles based on E2 Protein and Evaluation of Its Immune Efficacy in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5143-5153.
[10]	HAN Shengyi, LI Lingxia, LI Shuping, HU Guoyuan, LI Shengqing. Biological Characteristics and Genome Analysis of Salmonella Phage SP3 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5228-5239.
[11]	LI Linyan, TENG Man, LIU Jinling, ZHENG Luping, CHAI Shujun, DING Ke, YU Zuhua, LUO Jun. Development of a Monoclonal Antibody Pool for Marek’s Disease Virus and Identification of a Specific mAb against the Glycoprotein gB [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4712-4723.
[12]	CEN Xin, YANG Tingting, ZHAO Zunfu, WEN Yongping, ZHANG Huanrong. Isolation, Identification, Biological Characteristics and Genome Analysis of a Virulent Salmonella Phage [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2677-2688.
[13]	CHEN Xiaojun, MA Jun, CHEN Xiongnan, LIANG Yifan, GAO Qi, HUANG Zhao, XU Runda, ZHENG Jiachen, ZHENG Zezhong, ZHANG Guihong, WANG Heng, GONG Lang. Identification of the B Cell Epitopes Recognized by a Monoclonal Antibody against the p30 Protein of African Swine Fever Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2239-2251.
[14]	CAO Li, CHENG Runan, WU Zhouhui, WANG Jiawei, WANG Yu, ZHANG Yonghong, WU Qingmin, WANG Zhen. Construction and Biological Characteristics of sapC Gene Deletion Strain of Salmonella Typhimurium [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2282-2289.
[15]	ZENG Jinhong, XU Qingyuan, SHA Weiping, WU Shuang, LI Yuan, XIN Jiuqing, WANG Xiumei. Development and Application of Monoclonal Antibodies against Mycoplasma mycoides subsp.Mycoides [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2290-2299.