猪IL-1β单克隆抗体制备及其抗炎症反应活性

doi:10.11843/j.issn.0366-6964.2025.02.037

摘要/Abstract

摘要：

IL-1β可介导炎症病理，阻断IL-1活性具有重要抗炎治疗作用。本研究通过构建猪IL-1β原核表达质粒，进行大肠杆菌表达和蛋白纯化，制备重组蛋白。采用细胞杂交瘤技术制备获得4株稳定分泌IL-1β单克隆抗体杂交瘤细胞株(1E2、2H3、5H3及7E7)；4株单抗腹水ELISA效价高达1∶1 024 000；5H3单抗识别的抗原表位为¹⁴⁹DLKREVVFCM¹⁵⁸；1E2、2H3和7E7单抗所识别的抗原表位为²⁰²KRYPKRD²⁰⁸。脂多糖(LPS)小鼠炎症模型抗炎试验结果显示，相对LPS对照组，IL-1β单抗处理组小鼠血清NO、TNF-α、IL-6和IL-1β含量及临床症状评分显著降低，表明4株单抗均有较好抗炎效果，其中1E2单抗抗炎作用最优，为猪IL-1β阻断药物研究奠定了重要基础。

关键词: IL-1β, 单克隆抗体, 炎症治疗

Abstract:

IL-1β mediates inflammatory pathology, and blocking IL-1 activity has important anti-inflammatory effects. In this study, recombinant protein was prepared by constructing porcine IL-1β prokaryotic expression plasmid, expression by Escherichia coli and protein purification. Four hybridoma cell lines (1E2, 2H3, 5H3 and 7E7) with stable IL-1β monoclonal antibody secretion were prepared by cell hybridoma technique. The ELISA titer of the 4 mAbs in ascites were up to 1: 1 024 000. The epitope of 5H3 mAb was ¹⁴⁹DLKREVVFCM¹⁵⁸. The epitope recognized by 1E2, 2H3 and 7E7 mAb was ²⁰²KRYPKRD²⁰⁸. The anti-inflammatory test results of lipopolysaccharide (LPS) mice inflammation model showed that compared with LPS control group, serum NO, TNF-α, IL-6 and IL-1β contents and clinical symptom score of mice treated with IL-1β monoclonal antibody were significantly reduced, indicating that all the four strains of monoclonal antibody had better anti-inflammatory effect, and 1E2 monoclonal antibody had the best anti-inflammatory effect. It laid an important foundation for the study of porcine IL-1β blocking drugs.

Key words: IL-1β, monoclonal antibody, inflammatory therapy

中图分类号:

S852.659.6

李璠, 孙海凤, 孙萌, 高雁怩, 孙杨杨, 张路捷, 白娟, 姜平. 猪IL-1β单克隆抗体制备及其抗炎症反应活性[J]. 畜牧兽医学报, 2025, 56(2): 890-899.

LI Fan, SUN Haifeng, SUN Meng, GAO Yanxiao, SUN Yangyang, ZHANG Lujie, BAI Juan, JIANG Ping. Preparation and Anti-inflammatory Activity of Porcine IL-1β Monoclonal Antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 890-899.

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引物名称 Primers	序列(5′→3′) Sequences	扩增区域/aa Region	基因长度/bp Gene length
F1	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$GTGTGGGCCAGCCAGCACTAGAG	115-138	72
F2	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$CCCTGTGAGGAGGTGGAGAGCC	115-148	102
F3	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$CATGCAGAACACCACTTCTCTCT	115-158	132
F4	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$GTTGTTGCTATCATCTCCTTGCAC	115-168	162
F5	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$TCCCTTGATCCCTAAGGTCACAG	115-178	192
F6	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$GGGGTCTATATCCTCCAGCTGCA	115-201	261
F7	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$GTCCCTCTTTGGGTATCTTTTGGG	115-208	282
F8	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$TGTCTTGTAGAAGACAAATCGCT	115-218	312
F9	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$AGACTCGAATTCAACTCTGTTCT	115-228	342
F10	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$GGTGCTGATGTACCAGTTGGGGT	115-238	372
F11	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$CAGGAAGACGGGCTTTTGTTCTG	115-248	402
F12	F: CGC$\underline{{\rm{GGATCC}}}$GCCAACGTGCAGTCTATGGAGTG R: CCG$\underline{{\rm{CTCGAG}}}$AGTTATATCTTGGCGGCCTTTGGA	115-258	432

序号 Serial number	参数 Parameter	评分标准 Evaluation criteria	分值 Score
1	外观	光泽，光滑	0
		部分毛发竖立	1
		背部全部毛发竖立	2
		蓬松，凌乱	3
		憔悴	4
2	自主意识	活跃	0
		活跃，不直立	1
		活动减少迟缓，仍然行走	2
		挑衅后才活动，行动震颤	3
		挑衅后仍静止不动	4
3	对刺激的反应	迅速反应	0
		对声音无反应或者反应慢，对触碰马上逃离	1
		对声音无反应，对触碰反应一般(走几步)	2
		对声音无反应，对触碰反应弱(不移动)	3
		对声音和触碰几乎无反应，推倒后不能翻身	4
4	眼睛	全开	0
		没有全开，可能有分泌物	1
		半闭，可能有分泌物	2
		没有全闭，可能有分泌物	3
		闭合或者显著分泌物(浑浊)	4
5	呼吸频率	正常	0
		轻微减少(肉眼无法计数)	1
		中度减少(肉眼勉强能计数)	2
		显著减少(肉眼能轻松计数，呼吸间隔0.5s)	3
		极度减少(呼吸间隔＞1s)	4
6	呼吸质量	正常	0
		间断性呼吸困难	1
		呼吸困难，不喘气	2
		呼吸困难，间断性喘气	3
		喘气	4

组别 Group	攻毒后不同时间点评分 Score at different time points after attack
组别 Group	3 h	6 h	9 h	12 h	24 h	36 h
空白组Blank	0^Cc	0^Cc	0^Cc	0^Cc	0^Cc	0 ^Bb
LPS对照组LPS control	5.80±1.64^Aa	7.80±1.64^Aa	12.60±0.55 ^Aa	15.20±045 ^Aa	15.60±2.50^Aa	18.60±5.86^Aa
地塞米松组Dexamethasone formation	2.20±1.10^BbC	2.80±2.45^BbCc	7.40±1.92^Bb	6.20±0.84 ^Bb	5.20±2.49^Bb	3.60±3.33 ^Bb
1E2单抗处理组1E2 mAb treatment	2.40±1.34 ^BbC	3.80±2.49^ABbc	8.60±2.91^ABb	8.00±2.55 ^Bb	7.00±2.34^Bb	4.00±2.12 ^Bb
2H3单抗处理组2H3 mAb treatment	3.40±1.34^ABb	5.20±2.39^AaBb	9.00±2.07 ^ABb	8.60±2.07 ^Bb	7.20±2.28^Bb	4.40±1.82 ^Bb

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