Summary

猪肠粘膜上皮中靶向氨基肽酶N的单克隆抗体的生产

Published: May 18, 2021
doi:

Summary

pIRES2-ZSGreen1-rAbs-APN-CHO细胞表达的重组抗体蛋白和采用传统杂交瘤技术生产的单克隆抗体可以识别并与猪氨基肽酶N(APN)蛋白结合。

Abstract

猪氨基肽酶N(APN)是一种大量存在于小肠粘膜中的膜结合金属肽酶,可以启动粘膜免疫反应,而不会受到任何干扰,例如低蛋白表达,酶缺乏活性或结构变化。这使得APN成为选择性靶向粘膜上皮的疫苗开发的一个有吸引力的候选者。先前的研究表明,APN是产肠毒素 肠杆菌(大肠杆菌)F4和传染性胃肠炎病毒的受体蛋白。因此,APN在开发基于APN特异性抗体的抗体-药物偶联物或新型疫苗方面显示出前景。在这项研究中,我们比较了使用传统杂交瘤技术和重组抗体表达方法生产的APN特异性单克隆抗体(mAb)。我们还使用pIRES2-ZSGreen1-rAbs-APN和含有pET28a (+)-rAbs-APN载体的大 肠杆菌 表达BL21(DE3)菌株建立了稳定转染的中国仓鼠卵巢(CHO)细胞系。结果表明,在pIRES2-ZSGreen1-rAbs-APN-CHO细胞中表达的抗体以及使用杂交瘤产生的mAb可以识别并与APN蛋白结合。这为进一步阐明APN受体功能提供了基础,以开发针对不同APN特异性表位的疗法。

Introduction

氨基肽酶 N (APN) 是一种属于金属蛋白酶 M1 家族的月光酶,通过酶依赖性和酶依赖性途径充当肿瘤标志物、受体和信号分子12。APN除了裂解各种生物活性肽的N端氨基酸残基以调节其生物活性外,在各种炎症性疾病的发病机制中起着重要作用。APN 通过与主要组织相容性复合物 II 类分子23 紧密结合的修剪肽参与抗原加工和呈递。APN 还通过与参与多种信号转导的 G 蛋白偶联受体结合、调节细胞因子分泌以及促进免疫应答中 Fc γ 受体介导的吞噬作用来发挥抗炎作用4567

作为一种广泛分布的膜结合外切肽酶,APN在猪小肠粘膜中含量丰富,与受体介导的内吞作用158密切相关。APN识别并结合传染性胃肠炎病毒的刺突蛋白进入细胞,并直接与产肠毒素大肠杆菌F4菌毛的FaeG亚基相互作用,影响细菌与宿主细胞91011的粘附。因此,APN是治疗病毒和细菌传染病的潜在治疗靶点。

自1975年开发杂交瘤技术和其他单克隆抗体(mAb)生产策略以来,mAb已广泛用于免疫治疗、药物递送和诊断121314。目前,mAb已成功用于治疗癌症、炎症性肠病和多发性硬化症等疾病1215。由于其强大的亲和力和特异性,mAb可以成为抗体-药物偶联物(ADC)或新疫苗开发的理想靶标1617。APN蛋白对于选择性地将抗原递送到特定细胞至关重要,并且可以引发针对病原体的特异性和强大的粘膜免疫反应,而不会受到任何干扰,包括低蛋白表达,酶无活性或结构变化5818因此,基于APN特异性单克隆抗体的治疗产品有望对抗细菌和病毒感染。在这项研究中,我们描述了使用杂交瘤技术生产APN特异性mAb,以及使用原核和真核载体表达抗APN重组抗体(rAb)。结果表明,pIRES2-ZSGreen1-rAbs-APN-CHO细胞和杂交瘤衍生的mAb均能识别APN蛋白。

Protocol

本研究中的所有动物实验均获得扬州大学机构动物护理和使用委员会(SYXK20200041)的批准。 1.猪APN蛋白抗原的制备 注意:pET28a (+)-APN-BL21 (DE3) 菌株和 APN 稳定表达的细胞 pEGFP-C1-APN-IPEC-J2 是在先前的研究11 中构建的。 从冷冻甘油原液中回收细菌,并划线到含有 50 μg/mL 卡那霉素 (Km+) 的 Luria-Bertani (LB) 平板上进行?…

Representative Results

在本研究中,纯化的可溶性APN蛋白(2.12 mg / mL)用于小鼠免疫。每隔14天接种APN蛋白四次的小鼠在其血清中表现出更高的APN抗体滴度。尽管使用融合实验获得了14个杂交瘤,但只有9个杂交瘤在三个连续冻融循环中幸存下来,从而产生了9个分泌APN抗体的稳定克隆。所有这些细胞都是圆形、明亮和透明的(图1)。经SDS-PAGE确认纯化的mAb具有重链和轻链(分别为50 kDa和25 kDa),并?…

Discussion

诱导粘膜免疫是对抗病原体和预防和治疗各种疾病的最有效方法之一。APN是肠粘膜中一种高表达的膜结合蛋白,参与诱导适应性免疫反应和受体介导的病毒和细菌内吞作用158。APN 在多种形式的抗原加载和疫苗递送中用作抗原颗粒。口服APN靶向抗体也可以引发有效的免疫反应1824</sup…

Declarações

The authors have nothing to disclose.

Acknowledgements

本研究得到了国家自然科学基金(31702242年第32072820号)、江苏省政府海外留学奖学金(JS20190246)和扬州大学科学研究基金高层次人才资助,该基金由江苏省高等学校发展优先学术项目资助。

Materials

Complete Freund’s adjuvant Sigma-Aldrich F5881 Animal immunization
DAPI Beyotime  Biotechnology C1002 Nuclear counterstain
DMEM Gibco 11965092 Cell culture
DMEM-F12 Gibco 12634010 Cell culture
Dylight 549-conjugated goat anti-mouse IgG secondary antibody Abbkine A23310 Indirect immunofluorescence analysis
Enhanced Cell Counting Kit-8 Beyotime  Biotechnology C0042 Measurement of cell viability and vitality
Fetal bovine serum Gibco 10091 Cell culture
Geneticin™ Selective Antibiotic Gibco 11811098 Selective antibiotic
HAT Supplement (50X) Gibco 21060017 Cell selection
HT Supplement (100X) Gibco 11067030 Cell selection
Incomplete Freund’s adjuvant Sigma-Aldrich F5506 Animal immunization
isopropyl β-d-1-thiogalactopyranoside Sigma-Aldrich I5502 Protein expression
kanamycin Beyotime  Biotechnology ST102 Bactericidal antibiotic
Leica TCS SP8 STED confocal microscope Leica Microsystems  SP8 STED Fluorescence imaging
Lipofectamine® 2000 Reagent Thermofisher 11668019 Transfection
LSRFortessa™ fluorescence-activated cell sorting BD FACS LSRFortessa Flow cytometry
Microplate reader BioTek BOX 998 ELISA analysis
Micro spectrophotometer Thermo Fisher Nano Drop one Nucleic acid concentration detection
NaCl Sinopharm Chemical Reagent 10019308 Culture broth
(NH4)2SO4 Sinopharm Chemical Reagent 10002917 Culture broth
Opti-MEM Gibco 31985088 Cell culture
Polyethylene glycol 1500 Roche Diagnostics 10783641001 Cell fusion
PrimeScript™ 1st strand cDNA Synthesis Kit Takara Bio RR047 qPCR
protein A agarose Beyotime  Biotechnology P2006 Antibody protein purification
Protino® Ni+-TED 2000 Packed Columns MACHEREY-NAGEL 745120.5 Protein purification
SBA Clonotyping System-HRP Southern Biotech May-00 Isotyping of mouse monoclonal antibodies
Seamless Cloning Kit Beyotime  Biotechnology D7010S Construction of plasmids
Shake flasks Beyotime  Biotechnology E3285 Cell culture
Sodium carbonate-sodium bicarbonate buffer Beyotime  Biotechnology C0221A Cell culture
Trans-Blot SD Semi-Dry Transfer Cell Bio-rad  170-3940 Western blot
Tryptone Oxoid LP0042 Culture broth
Ultrasonic Homogenizer Ningbo Xinzhi Biotechnology JY92-IIN Sample homogenization
Yeast extract Oxoid LP0021 Culture broth
96-well microplate Corning 3599 Cell culture

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Xia, P., Lian, S., Wu, Y., Yan, L. Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium. J. Vis. Exp. (171), e62437, doi:10.3791/62437 (2021).

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