Name: production of monoclonal antibodies targeting aminopeptidase n in the porcine intestinal mucosal epithelium
Uploaded: 2021-05-18T13:00:00
Description: Watch this Scientific Journal Video about Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium at JoVE.com

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.

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.
<ol>
	<li>Recover bacteria from a frozen glycerol stock and streak onto Luria-Bertani (LB) plates containing 50 &#181;g/m...

<ol>
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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...

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 molecules2,3. APN also exerts anti-inflammatory effects by binding with G protein-coupled receptors participating in multiple signal transduction, modulating cytokine secretion, and contributing to Fc gamma receptor-mediated phagocytosis in the immune response4,5,6,7.
As a widely distributed membrane-bound exopeptidase, APN is abundant in the porcine small intestinal mucosa and is closely associated with receptor-mediated endocytosis1,5,8. APN recognizes and binds the spike protein of the transmissible gastroenteritis virus for cell entry, and directly interacts with the FaeG subunit of enterotoxigenic Escherichia coli F4 fimbriae to affect bacterial adherence with host cells9,10,11. Thus, APN is a potential therapeutic target in the treatment of viral and bacterial infectious diseases.
Since the development of hybridoma technology and other strategies for monoclonal antibodies (mAbs) production in 1975, mAbs have been widely used in immunotherapy, drug delivery, and diagnosis12,13,14. Currently, mAbs are successfully used to treat diseases, such as cancer, inflammatory bowel disease, and multiple sclerosis12,15. Because of their strong affinity and specificity, mAbs can be ideal targets in the development of antibody-drug conjugates (ADC) or new vaccines16,17. The APN protein is critical for selectively delivering antigens to specific cells, and can elicit a specific and strong mucosal immune response against pathogens without any interference including low protein expression, enzyme inactivity, or structural changes5,8,18. Therefore, therapeutic products based on APN-specific mAbs show promise against bacterial and viral infections. In this study, we describe the production of APN-specific mAbs using hybridoma technology, and expression of anti-APN recombinant antibodies (rAbs) using prokaryotic and eukaryotic vectors. The result indicates that the APN protein was recognized by both rAbs expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and hybridoma-derived mAbs.

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		<tr>
			<td>Complete Freund&#8217;s adjuvant</td>
			<td>Sigma-Aldrich</td>
			<td>F5881</td>
			<td>Animal immunization</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>C1002</td>
			<td>Nuclear counterstain</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td>Cell culture</td>
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		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Dylight 549-conjugated goat anti-mouse IgG secondary antibody</td>
			<td>Abbkine</td>
			<td>A23310</td>
			<td>Indirect immunofluorescence analysis</td>
		</tr>
		<tr>
			<td>Enhanced Cell Counting Kit-8</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>C0042</td>
			<td>Measurement&#160;of&#160;cell&#160;viability and vitality</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10091</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Geneticin&#8482; Selective Antibiotic</td>
			<td>Gibco</td>
			<td>11811098</td>
			<td>Selective antibiotic</td>
		</tr>
		<tr>
			<td>HAT Supplement (50X)</td>
			<td>Gibco</td>
			<td>21060017</td>
			<td>Cell selection</td>
		</tr>
		<tr>
			<td>HT Supplement (100X)</td>
			<td>Gibco</td>
			<td>11067030</td>
			<td>Cell selection</td>
		</tr>
		<tr>
			<td>Incomplete Freund&#8217;s adjuvant</td>
			<td>Sigma-Aldrich</td>
			<td>F5506</td>
			<td>Animal immunization</td>
		</tr>
		<tr>
			<td>isopropyl &#946;-d-1-thiogalactopyranoside</td>
			<td>Sigma-Aldrich</td>
			<td>I5502</td>
			<td>Protein expression</td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>ST102</td>
			<td>Bactericidal antibiotic</td>
		</tr>
		<tr>
			<td>Leica TCS SP8 STED confocal microscope</td>
			<td>Leica Microsystems</td>
			<td>&#160;SP8 STED</td>
			<td>Fluorescence imaging</td>
		</tr>
		<tr>
			<td>Lipofectamine&#174; 2000 Reagent</td>
			<td>Thermofisher</td>
			<td>11668019</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>LSRFortessa&#8482; fluorescence-activated cell sorting</td>
			<td>BD</td>
			<td>FACS LSRFortessa</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>BOX 998</td>
			<td>ELISA analysis</td>
		</tr>
		<tr>
			<td>Micro spectrophotometer</td>
			<td>Thermo Fisher</td>
			<td>Nano Drop one</td>
			<td>Nucleic acid concentration detection</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019308</td>
			<td>Culture broth</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10002917</td>
			<td>Culture broth</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985088</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Polyethylene glycol 1500</td>
			<td>Roche Diagnostics</td>
			<td>10783641001</td>
			<td>Cell fusion</td>
		</tr>
		<tr>
			<td>PrimeScript&#8482; 1st strand cDNA Synthesis Kit</td>
			<td>Takara Bio</td>
			<td>RR047</td>
			<td>qPCR</td>
		</tr>
		<tr>
			<td>protein A agarose</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>P2006</td>
			<td>Antibody protein purification</td>
		</tr>
		<tr>
			<td>Protino&#174; Ni+-TED 2000 Packed Columns</td>
			<td>MACHEREY-NAGEL</td>
			<td>745120.5</td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>SBA Clonotyping System-HRP</td>
			<td>Southern Biotech</td>
			<td>May-00</td>
			<td>Isotyping of mouse monoclonal antibodies</td>
		</tr>
		<tr>
			<td>Seamless Cloning Kit</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>D7010S</td>
			<td>Construction of plasmids</td>
		</tr>
		<tr>
			<td>Shake flasks</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>E3285</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Sodium carbonate-sodium bicarbonate buffer</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>C0221A</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-rad</td>
			<td>&#160;170-3940</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>Culture broth</td>
		</tr>
		<tr>
			<td>Ultrasonic Homogenizer</td>
			<td>Ningbo Xinzhi Biotechnology</td>
			<td>JY92-IIN</td>
			<td>Sample homogenization</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td>Culture broth</td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Corning</td>
			<td>3599</td>
			<td>Cell culture</td>
		</tr>
	</tbody>
</table>

production of monoclonal antibodies targeting aminopeptidase n in the porcine intestinal mucosal epithelium

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.

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...

Watch this Scientific Journal Video about Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium at JoVE.com

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

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 ...

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Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...