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Immunology and Infection

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Published: May 18th, 2021

DOI:

10.3791/62437

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

The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

Aminopeptidase N (APN), a moonlighting enzyme that belongs to the metalloproteinase M1 family, acts as a tumor marker, receptor, and signaling molecule via enzyme-dependent and enzyme-independent pathways1,2. In addition to cleaving the N-terminal amino-acid residues of various bioactive peptides for the regulation of their biological activity, APN plays an important role in the pathogenesis of various inflammatory diseases. APN participates in antigen processing and presentation by trimmed peptides that bind tightly to major histocompatibility complex class II molecules2,

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All animal experiments in this study were approved by the Yangzhou University Institutional Animal Care and Use Committee (SYXK20200041).

1. Preparation of porcine APN protein antigen

NOTE: The pET28a (+)-APN-BL21 (DE3) strain and the APN stably expressed cells pEGFP-C1-APN-IPEC-J2 were constructed in a previous study11.

  1. Recover bacteria from a frozen glycerol stock and streak onto Luria-Bertani (LB) plates containing 50 µg/m.......

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In this study, the purified soluble APN protein (2.12 mg/mL) was used for mouse immunization. Mice immunized with the APN protein four times at 14-day intervals exhibited a higher antibody titer against APN in their sera. Although 14 hybridomas were obtained using the fusion experiments, only 9 hybridomas survived the three continuous freeze-thaw cycles, resulting in 9 stable clones that secreted antibodies against APN. All these cells are round, bright, and clear (Figure 1). The purified mA.......

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Induction of mucosal immunity is one of the most effective approaches in counteracting pathogens and in prevention and treatment of various diseases. APN, a highly expressed membrane-bound protein in the intestinal mucosa, is involved in the induction of adaptive immune response and in receptor-mediated viral and bacterial endocytosis1,5,8. APN is used as antigen particulate in many formats of antigen loading and vaccine deliver.......

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This study was supported by the Chinese National Science Foundation Grant (No. 32072820, 31702242), grants from Jiangsu Government Scholarship for Overseas Studies (JS20190246) and High-level Talents of Yangzhou University Scientific Research Foundation, a project founded by the Priority Academic Program of Development Jiangsu High Education Institution.

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Name Company Catalog Number Comments
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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