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

Summary

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.

Abstract

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.

Introduction

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 pathways^1,2. 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 molecules^2,

Protocol

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.......

Discussion

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 endocytosis^1,5,8. APN is used as antigen particulate in many formats of antigen loading and vaccine deliver.......

Materials

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