猪肠粘膜上皮中靶向氨基肽酶N的单克隆抗体的生产
1College of Veterinary Medicine (Institute of comparative medicine),Yangzhou University, 2Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, 3Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China,Yangzhou University
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 大 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1，2。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2，3 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4，5，6，7。
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1，5，8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9，10，11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12，13，14。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12，15。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16，17。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5，8，18。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1，5，8。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18，24</sup…
Divulgazioni
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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Citazione di questo articolo
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).