一株猪CD56单克隆抗体的制备及抗原表位的初步鉴定

doi:10.11843/j.issn.0366-6964.2024.06.037

Abstract

Abstract:

As an important surface molecule of NK cells, CD56 plays an important role in the development, subgroup typing and function of NK cells. Therefore, in order to facilitate the screening of different subpopulations of porcine NK cells and explore their biological functions, this study prepared porcine CD56 monoclonal antibodies and preliminically identified their epitopes. Firstly, the amino acid sequences of the dominant epitope of porcine CD56 protein extracellular region (50-499aa) was predicted and selected by bioinformatics software. Then the selected region of CD56 was expressed by prokaryotic expression system, and the protein was purified and renaturated. After that, the recombinant protein was immunized to BALB/c mice, and a hybridoma cell line with stable secretion of porcine CD56 monoclonal antibody was obtained through cell fusion and subcloning experiments, which was named 1G8. Western blot results showed that the prepared porcine CD56 monoclonal antibody could bind specifically to the total protein of porcine brain and spleen. To further explore the specificity of CD56 monoclonal antibody, NK cells were isolated from porcine peripheral blood, then indirect immunofluorescence assay (IFA) was performed by using the prepared CD56 monoclonal antibody as the primary antibody. The results showed that the prepared monoclonal antibody could bind specifically to NK cell membrane. Finally, in order to further investigate the epitopes recognized by monoclonal antibodies, we further truncated the selected porcine CD56 gene into four truncations (50aa-192aa, 193aa-294aa, 295aa-397aa, 398aa-499aa), which were insert into eukaryotic expression plasmids. And then these eukaryotic expression recombinant plasmids were transfected into 293T cells, respectively. Western blotting and IFA results showed that the epitopes recognized by the prepared CD56 monoclonal antibody were 193aa-294aa of porcine CD56 protein. In this study, porcine CD56 monoclonal antibody was successfully prepared, and its specificity and epitopes were preliminically identified, which is of great significance for screening porcine NK cell subsets and exploring the biological function of porcine NK cells

Key words: pig, NK cells, CD56, monoclonal antibody, epitope

CLC Number:

S852.4

Hangkuo XIN, Qingqing XIE, Kun LIU, Shengyu LIN, Wei CHEN, Xiaohui ZHENG, Ting ZHU. Preparation of Monoclonal Antibody against Porcine CD56 and Preliminary Identification of Its Epitope[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2662-2671.

Figures/Tables 9

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References 26

1	MYERS J A , MILLER J S . Exploring the NK cell platform for cancer immunotherapy[J]. Nat Rev Clin Oncol, 2021, 18 (2): 85- 100. doi: 10.1038/s41571-020-0426-7
2	张义伟, 苏紫薇, 李其龙, 等. 伯氏疟原虫ANKA株感染小鼠的T细胞、NK细胞及细胞因子变化[J]. 畜牧兽医学报, 2022, 53 (11): 4008- 4018.
	ZHANG Y W , SU Z W , LI Q L , et al. The changes of T cells, NK cells and Cytokines in mice infected with plasmodium berghei ANKA strain[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53 (11): 4008- 4018.
3	WAGGONER S N , REIGHARD S D , GYUROVA I E , et al. Roles of natural killer cells in antiviral immunity[J]. Curr Opin Virol, 2016, 16, 15- 23. doi: 10.1016/j.coviro.2015.10.008
4	AYALA-NUNEZ N V , FOLLAIN G , DELALANDE F , et al. Zika virus enhances monocyte adhesion and transmigration favoring viral dissemination to neural cells[J]. Nat Commun, 2019, 10 (1): 4430. doi: 10.1038/s41467-019-12408-x
5	HOTTA K , MOTOI Y , OKUTANI A , et al. Role of GPI-anchored NCAM-120 in rabies virus infection[J]. Microbes Infect, 2007, 9 (2): 167- 174. doi: 10.1016/j.micinf.2006.11.003
6	YANG A H , CHEN J Y , LEE C H , et al. Expression of NCAM and OCIAD1 in well-differentiated thyroid carcinoma: correlation with the risk of distant metastasis[J]. J Clin Pathol, 2012, 65 (3): 206- 212. doi: 10.1136/jclinpath-2011-200416
7	SCHREIBER S C , GIEHL K , KASTILAN C , et al. Polysialylated NCAM represses E-cadherin-mediated cell-cell adhesion in pancreatic tumor cells[J]. Gastroenterology, 2008, 134 (5): 1555- 1566. doi: 10.1053/j.gastro.2008.02.023
8	李湘青, 殷欣, 赵雪莲, 等. NK细胞亚群CD56^bright在帕金森患者外周血中的表达及临床意义[J]. 山东大学学报: 医学版, 2021, 59 (2): 34- 40.
	LI X Q , YIN X , ZHAO X L , et al. Expression and clinical significance of circulating CD56^bright subset of NK cells in patients with Parkinson's disease[J]. Journal of Shandong University: Health Sciences, 2021, 59 (2): 34- 40.
9	FREUD A G , MUNDY-BOSSE B L , YU J H , et al. The broad spectrum of human natural killer cell diversity[J]. Immunity, 2017, 47 (5): 820- 833. doi: 10.1016/j.immuni.2017.10.008
10	PICARD L K , CLAUS M , FASBENDER F , et al. Human NK cells responses are enhanced by CD56 engagement[J]. Eur J Immunol, 2022, 52 (9): 1441- 1451. doi: 10.1002/eji.202249868
11	DE PELSMAEKER S , DIERICK E , KLUPP B , et al. Expression of the pseudorabies virus gB glycoprotein triggers NK cell cytotoxicity and increases binding of the activating NK cell receptor PILRβ[J]. J Virol, 2019, 93 (7): e02107- 18.
12	GRAUWET K , CANTONI C , PARODI M , et al. Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1-dependent NK cell-mediated lysis of infected cells[J]. Proc Natl Acad Sci U S A, 2014, 111 (45): 16118- 16123. doi: 10.1073/pnas.1409485111
13	GRAUWET K , VITALE M , DE PELSMAEKER S , et al. Pseudorabies virus US3 protein kinase protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a[J]. J Virol, 2016, 90 (3): 1522- 1533. doi: 10.1128/JVI.02902-15
14	DASSA L , SEIDEL E , OIKNINE-DJIAN E , et al. The human cytomegalovirus protein UL148A downregulates the NK cell-activating ligand MICA to avoid NK cell attack[J]. J Virol, 2018, 92 (17): e00162- 18.
15	SMITH D W , CHAKRAVORTY A , HAYES M , et al. The epstein-barr virus oncogene EBNA1 suppresses natural killer cell responses and apoptosis early after infection of peripheral B cells[J]. mBio, 2021, 12 (6): e02243- 21.
16	ZHENG J , WEN L Y , YEN H L , et al. Phenotypic and functional characteristics of a novel influenza virus hemagglutinin-specific memory NK cell[J]. J Virol, 2021, 95 (12): e00165- 21.
17	POLI A , MICHEL T , THÉRÉSINE M , et al. CD56^bright natural killer (NK) cells: an important NK cell subset[J]. Immunology, 2009, 126 (4): 458- 465. doi: 10.1111/j.1365-2567.2008.03027.x
18	LEE S , WATSON M W , FLEXMAN J P , et al. Increased proportion of the CD56^bright NK cell subset in patients chronically infected with hepatitis C virus (HCV) receiving interferon-αand ribavirin therapy[J]. J Med Virol, 2010, 82 (4): 568- 574. doi: 10.1002/jmv.21742
19	ZHANG Y Z , TONG S W , LI S Y , et al. Increased ILT2 expression contributes to dysfunction of CD56^dimCD16⁺NK cells in chronic hepatitis B virus infection[J]. Antiviral Res, 2022, 205, 105385. doi: 10.1016/j.antiviral.2022.105385
20	JIANG Y J , CHEN Y X , CHEN L L , et al. Impaired circulating CD56^dim NK cells are associated with decompensation of HBV-related cirrhosis[J]. Hum Immunol, 2020, 81 (1): 32- 40. doi: 10.1016/j.humimm.2019.11.006
21	MACE E M , GUNESCH J T , DIXON A , et al. Human NK cell development requires CD56-mediated motility and formation of the developmental synapse[J]. Nat Commun, 2016, 7, 12171. doi: 10.1038/ncomms12171
22	NITTA T , YAGITA H , SATO K , et al. Involvement of CD56 (NKH-1/Leu-19 antigen) as an adhesion molecule in natural killer-target cell interaction[J]. J Exp Med, 1989, 170 (5): 1757- 1761. doi: 10.1084/jem.170.5.1757
23	GUNESCH J T , DIXON A L , EBRAHIM T , et al. CD56 regulates human NK cell cytotoxicity through Pyk2[J]. eLife, 2020, 9, e57346. doi: 10.7554/eLife.57346
24	THOULOUZE M I , LAFAGE M , SCHACHNER M , et al. The neural cell adhesion molecule is a receptor for rabies virus[J]. J Virol, 1998, 72 (9): 7181- 7190. doi: 10.1128/JVI.72.9.7181-7190.1998
25	SRIVASTAVA M , ZHANG Y , CHEN J , et al. Chemical proteomics tracks virus entry and uncovers NCAM1 as Zika virus receptor[J]. Nat Commun, 2020, 11 (1): 3896. doi: 10.1038/s41467-020-17638-y
26	李俊慧, 邵军军, 常惠芸. 抗原表位鉴定方法的研究进展[J]. 中国兽医科学, 2021, 51 (6): 678- 683.
	LI J H , SHAO J J , CHANG H Y . Advance of antigenic epitope mapping methods[J]. Chinese Veterinary Science, 2021, 51 (6): 678- 683.

截短体 Truncated body	方向 Orientation	序列信息(5′ →3′) Sequence of information
Δ1	Forward	CCCAAGCTTCCGACATCTCCTGGTTCTCCCCC
	Reverse	CGCGGATCCCCTGCCCTCACAGCG
Δ2	Forward	CCCAAGCTTCCATCCTGGCCCGGGGG
	Reverse	CGCGGATCCCTCGCCGGCCTTGTTCT
Δ3	Forward	CCCAAGCTTCCCAGGATGCGTCCATCCATCTCA
	Reverse	CGCGGATCCGGCCGTGCAGACGTACT
Δ4	Forward	CCCAAGCTTCCAGCAACACCATCGGCCAGG
	Reverse	CGCGGATCCCAGGGACTCCTGTCCAATGCG

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Preparation of Monoclonal Antibody against Porcine CD56 and Preliminary Identification of Its Epitope

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