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

摘要/Abstract

摘要：

CD56作为NK细胞的重要表面分子，在NK细胞的发育、亚群分型和功能发挥等方面发挥重要的作用。因此，为了便于筛选不同亚群的猪NK细胞并探究其生物学功能，本研究制备了猪CD56单克隆抗体并对其抗原表位进行了初步鉴定。首先，通过生物信息软件预测并选取猪CD56蛋白胞外区的优势抗原表位的氨基酸序列(50-499aa)，利用原核表达系统表达猪CD56胞外区的优势抗原表位蛋白并进行纯化和复性，之后，将猪CD56重组蛋白免疫BALB/c小鼠，通过细胞融合、亚克隆等试验，获得一株稳定分泌猪CD56单克隆抗体的杂交瘤细胞株，命名为1G8。Western blot结果显示，制备的猪CD56单抗可以与猪脑和脾总蛋白发生特异性结合；为了进一步验证CD56单抗的特异性，作者分离了猪外周血NK细胞，以制备的猪CD56单抗作为一抗进行间接免疫荧光试验，结果显示，制备的单抗能够与NK细胞膜特异性结合。最后，为了进一步探究单抗识别的抗原表位，作者将选取的猪CD56基因进一步截短为四个截短体(50-192 aa、193-294 aa、295-397 aa、398-499 aa)并构建真核表达重组质粒，然后转染至293T细胞进行真核表达，Western blot和间接免疫荧光结果显示，制备的CD56单抗识别的抗原表位为猪CD56蛋白的193-294 aa。综上，本研究成功制备了猪CD56单克隆抗体，并对其特异性及抗原表位进行了初步鉴定，对猪NK细胞亚群的筛选及探究猪NK细胞的生物学功能具有重要意义。

关键词: 猪, NK细胞, CD56, 单克隆抗体, 抗原表位

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

中图分类号:

S852.4

辛航阔, 谢青青, 刘坤, 林圣宇, 陈炜, 郑小惠, 朱婷. 一株猪CD56单克隆抗体的制备及抗原表位的初步鉴定[J]. 畜牧兽医学报, 2024, 55(6): 2662-2671.

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.

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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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