Immunology and Infection
Published: May 18th, 2021
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,
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.
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.......
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.......
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.....
|Complete Freund’s adjuvant
|Dylight 549-conjugated goat anti-mouse IgG secondary antibody
|Indirect immunofluorescence analysis
|Enhanced Cell Counting Kit-8
|Measurement of cell viability and vitality
|Fetal bovine serum
|Geneticin™ Selective Antibiotic
|HAT Supplement (50X)
|HT Supplement (100X)
|Incomplete Freund’s adjuvant
|Leica TCS SP8 STED confocal microscope
|Lipofectamine® 2000 Reagent
|LSRFortessa™ fluorescence-activated cell sorting
|Nano Drop one
|Nucleic acid concentration detection
|Sinopharm Chemical Reagent
|Sinopharm Chemical Reagent
|Polyethylene glycol 1500
|PrimeScript™ 1st strand cDNA Synthesis Kit
|protein A agarose
|Antibody protein purification
|Protino® Ni+-TED 2000 Packed Columns
|SBA Clonotyping System-HRP
|Isotyping of mouse monoclonal antibodies
|Seamless Cloning Kit
|Construction of plasmids
|Sodium carbonate-sodium bicarbonate buffer
|Trans-Blot SD Semi-Dry Transfer Cell
|Ningbo Xinzhi Biotechnology
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