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

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。

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。
<ol><li>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?...

<ol>
	<li>Chen, L., Lin, Y. L., Peng, G., Li, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+multifunctional+roles+of+mammalian+aminopeptidase+N.">Structural basis for multifunctional roles of mammalian aminopeptidase N.</a> Proceedings of the National Academy of Sciences of The United States Of America. 109 (44), 17966-17971 (2012).</li><li>Mina-Osorio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+moonlighting+enzyme+CD13:+old+and+new+functions+to+target.">The moonlighting enzyme CD13: old and new functions to target.</a> Trends in Molecular Medicine. 14 (8), 361-371 (2008).</li><li>Lu, C., Amin, M. A., Fox, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD13/Aminopeptidase+N+is+a+potential+therapeutic+target+for+inflammatory+disorders.">CD13/Aminopeptidase N is a potential therapeutic target for inflammatory disorders.</a> The Journal of Immunology. 204 (1), 3-11 (2020).</li><li>Villase&#241;or-Cardoso, M. I., Frausto-Del-R&#237;o, D. A., Ortega, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminopeptidase+N+(CD13)+is+involved+in+phagocytic+processes+in+human+dendritic+cells+and+macrophages.">Aminopeptidase N (CD13) is involved in phagocytic processes in human dendritic cells and macrophages.</a> BioMed Research International. 2013, 562984 (2013).</li><li>Melkebeek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+aminopeptidase+N,+a+newly+identified+receptor+for+F4ac+fimbriae,+enhances+the+intestinal+mucosal+immune+response.">Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response.</a> Mucosal Immunology. 5 (6), 635-645 (2012).</li><li>Morgan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+function+of+aminopeptidase+N/CD13+produced+by+fibroblast-like+synoviocytes+in+rheumatoid+arthritis:+role+of+CD13+in+chemotaxis+of+cytokine-activated+T+cells+independent+of+enzymatic+activity.">Expression and function of aminopeptidase N/CD13 produced by fibroblast-like synoviocytes in rheumatoid arthritis: role of CD13 in chemotaxis of cytokine-activated T cells independent of enzymatic activity.</a> Arthritis &amp; Rheumatology. 67 (1), 74-85 (2015).</li><li>Du, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+and+arthritogenic+properties+of+the+soluble+form+of+CD13.">Angiogenic and arthritogenic properties of the soluble form of CD13.</a> The Journal of Immunology. 203 (2), 360-369 (2019).</li><li>Rasschaert, K., Devriendt, B., Favoreel, H., Goddeeris, B. M., Cox, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-mediated+endocytosis+and+transcytosis+of+enterotoxigenic+Escherichia+coli+F4+fimbriae+in+porcine+intestinal+epithelial+cells.">Clathrin-mediated endocytosis and transcytosis of enterotoxigenic Escherichia coli F4 fimbriae in porcine intestinal epithelial cells.</a> Veterinary Immunology and Immunopathology. 137 (3-4), 243-250 (2010).</li><li>Reguera, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bases+of+coronavirus+attachment+to+host+aminopeptidase+N+and+its+inhibition+by+neutralizing+antibodies.">Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies.</a> PLoS Pathogens. 8 (8), 100859 (2012).</li><li>Delmas, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminopeptidase+N+is+a+major+receptor+for+the+entero-pathogenic+coronavirus+TGEV.">Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV.</a> Nature. 357 (6377), (1992).</li><li>Xia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+aminopeptidase+N+binds+to+F4++enterotoxigenic+Escherichia+coli+fimbriae.">Porcine aminopeptidase N binds to F4+ enterotoxigenic Escherichia coli fimbriae.</a> Veterinary Research. 47 (1), 24 (2016).</li><li>Nakamura, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies:+methods+and+clinical+laboratory+applications.">Monoclonal antibodies: methods and clinical laboratory applications.</a> Clinical Physiology and Biochemistry. 1 (2-5), 160-172 (1983).</li><li>Chan, C. E., Chan, A. H., Lim, A. P., Hanson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+efficiency+of+antibody+selection+from+semi-synthetic+scFv+and+non-immune+Fab+phage+display+libraries+against+protein+targets+for+rapid+development+of+diagnostic+immunoassays.">Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.</a> Journal of Immunological Methods. 373 (1-2), 79-88 (2011).</li><li>K&#246;hler, G., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cultures+of+fused+cells+secreting+antibody+of+predefined+specificity.">Continuous cultures of fused cells secreting antibody of predefined specificity.</a> Nature. 256 (5517), 495-497 (1975).</li><li>El Miedany, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MABS:+targeted+therapy+tailored+to+the+patient's+need.">MABS: targeted therapy tailored to the patient's need.</a> British Journal of Nursing. 24 (16), 4-13 (2015).</li><li>Castelli, M. S., McGonigle, P., Hornby, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharmacology+and+therapeutic+applications+of+monoclonal+antibodies.">The pharmacology and therapeutic applications of monoclonal antibodies.</a> Pharmacology Research &amp; Perspectives. 7 (6), 00535 (2019).</li><li>Weiner, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+better+monoclonal+antibody-based+therapeutics.">Building better monoclonal antibody-based therapeutics.</a> Nature Reviews Cancer. 15 (6), 361-370 (2015).</li><li>Bakshi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+single-domain+antibodies+as+carriers+for+targeted+vaccine+delivery+to+the+small+intestinal+epithelium.">Evaluating single-domain antibodies as carriers for targeted vaccine delivery to the small intestinal epithelium.</a> Journal of Controlled Release. 321, 416-429 (2020).</li><li>Kohler, G., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cultures+of+fused+cells+secreting+antibody+of+predefined+specificity.">Continuous cultures of fused cells secreting antibody of predefined specificity.</a> The Journal of Immunology. 174 (5), 2453-2455 (2005).</li><li>Chen, W., Liu, W. E., Li, Y. M., Luo, S., Zhong, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+preliminary+application+of+monoclonal+antibodies+to+the+receptor+binding+region+of+Clostridium+difficile+toxin+B.">Preparation and preliminary application of monoclonal antibodies to the receptor binding region of Clostridium difficile toxin B.</a> Molecular Medicine Reports. 12 (5), 7712-7720 (2015).</li><li>Levieux, D., Venien, A., Levieux, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitopic+analysis+and+quantification+of+bovine+myoglobin+with+monoclonal+antibodies.">Epitopic analysis and quantification of bovine myoglobin with monoclonal antibodies.</a> Hybridoma. 14 (5), 435-442 (1995).</li><li>Zhou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellin+and+F4+fimbriae+have+opposite+effects+on+biofilm+formation+and+quorum+sensing+in+F4ac++enterotoxigenic+Escherichia+coli.">Flagellin and F4 fimbriae have opposite effects on biofilm formation and quorum sensing in F4ac+ enterotoxigenic Escherichia coli.</a> Veterinary Microbiology. 168 (1), 148-153 (2014).</li><li>Heinrich, L., Tissot, N., Hartmann, D. J., Cohen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+results+obtained+by+ELISA+and+surface+plasmon+resonance+for+the+determination+of+antibody+affinity.">Comparison of the results obtained by ELISA and surface plasmon resonance for the determination of antibody affinity.</a> Journal of Immunological Methods. 352 (1-2), 13-22 (2010).</li><li>Vander Weken, H., Cox, E., Devriendt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+oral+subunit+vaccine+design.">Advances in oral subunit vaccine design.</a> Vaccines. 9, 1 (2020).</li><li>Baert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-glucan+microparticles+targeted+to+epithelial+APN+as+oral+antigen+delivery+system.">&#946;-glucan microparticles targeted to epithelial APN as oral antigen delivery system.</a> Journal of Controlled Release. 220, 149-159 (2015).</li><li>Neuberger, M. S., Williams, G. T., Fox, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+antibodies+possessing+novel+effector+functions.">Recombinant antibodies possessing novel effector functions.</a> Nature. 312 (5995), 604-608 (1984).</li></ol>

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<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>
		</tr>
		<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

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。

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?...

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

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。

猪肠粘膜上皮中靶向氨基肽酶N的单克隆抗体的生产

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response ...

衍生单克隆抗体主要用于诊断治疗性免疫试剂，强调需要在有限的生产中生产稳定可靠的抗体。本文描述的方法证明，重组抗体生产可以提高单克隆抗体生产效率，并最大限度地降低人工和时间相关成本。这些方法用于开发氨基肽酶和基于抗体的抗体药物偶联物和其他治疗产品，这有助于阐明APN在预防和治疗细菌和病毒感染中的作用。演示该程序的是我实验室的研究生Yan Li。首先，腹膜内将100微克APN蛋白注射到选定的成年雌性小鼠中以进行最终抗原增强。注射三天后，收集小鼠的脾脏并用DMEM洗涤两次以去除血液和脂肪细胞。使用200目铜网格过滤脾细胞悬液以去除组织碎片，并使用离心法收获脾细胞以去除脾膜。将小鼠SP20骨髓瘤细胞接种在含有5毫升补充有6%胎牛血清的DMEM的25平方厘米烧瓶中，并在37摄氏度和6%二氧化碳气氛下培养细胞以维持细胞活力。培养五到六天后，复苏后细胞应达到80%至90%汇合，并在显微镜下呈现圆形，明亮和清晰。杂交前一天，从小鼠的腹膜腔收集巨噬细胞。在96孔板中以0.1至0.2乘以每毫升10至5倍的密度接种腹膜巨噬细胞，每个含有100微升HAT培养基并孵育过夜。对于杂交，用移液器轻轻吸出8至10瓶的SP20细胞，并将其悬浮在10毫升无血清DMEM培养基中。用新鲜的DMEM洗涤细胞并离心两次，然后将它们重悬于10毫升DMEM中。将定量的脾脏细胞与SB20细胞以10：1的比例混合，并将它们转移到50毫升管中。离心后，弃去上清液并收集管底部的沉淀。在杂交前轻拍试管以松开沉淀。使用滴管，在 45 秒内将一毫升预热至 37 摄氏度的聚乙二醇 1500 滴加到松散的细胞沉淀中，同时轻轻旋转管底部。然后将一毫升预热至37摄氏度的DMEM缓慢加入混合物中90秒，然后再加入30毫升新鲜DMEM，并将融合管放入37摄氏度的水浴中30分钟。孵育后，收获细胞。重悬HAT培养基并在接种有腹膜巨噬细胞的96孔板中培养。五天后，向每个孔中加入100微升新鲜HAT培养基。孵育五天后，再次用HAT培养基替换培养基。使用涂有 5 μg/毫升 APN 蛋白的微量滴定板，在 0.05 摩尔 PBS 中稀释，使用 ELISA 测定法分析杂交瘤上清液中的单克隆抗体。在 0.4 毫摩尔 β-d-1-吡喃半乳糖苷存在下，在 37 摄氏度的轨道振荡器中培养 pET28a 加重组抗体氨基肽酶 NBL21 转化细菌 10 小时。将每孔100微升0.5乘以10至第五个中国仓鼠卵巢细胞接种到96孔板中，在37摄氏度和6%二氧化碳气氛下孵育18至24小时。当细胞达到 80% 至 90% 汇合时，用 Opti-MEM 稀释 pIRES2-zs Green One 重组抗体氨基肽酶和质粒至终浓度为 0.1 微克/微升。五分钟后，将 50 微升稀释的 pIRES2-zs Green 一重组抗体氨基肽酶和质粒与 1 微升脂质胺 2000 和 49 微升 Opti-MEM 混合。孵育20分钟后，向含有CHO细胞的96孔板的每个孔中加入100微升混合物。在转染后4至6小时，用补充有10%FBS的DMEM F12替换培养基。孵育48小时后，向每个孔中加入400微克/微升G418，以选择稳定转染的细胞。使用补充有 10%FBS 和 400 微克/微升 G418 的 DMEM F12 培养基选择 10 天后，用荧光激活细胞分选对细胞进行分选。连续稀释收获的阳性细胞。在96孔板中以平均每孔0.5至2个细胞接种，并在37摄氏度，6%二氧化碳培养箱中培养。在代表性的显微镜分析中，所有杂交瘤细胞都呈现圆形、明亮和透明。经SDS-PAGE证实，纯化的单克隆抗体具有重50千道尔顿链和轻25千道尔顿链，并在纯化的腹水中发现。此处显示了培养物上清液和腹水中这些抗APN单克隆抗体的滴度。小鼠单克隆抗体同种分型显示，来源于克隆5B31、5B36、3C48、5C51和6C56的抗体具有免疫球蛋白G2B亚类，而APN 2A20为免疫球蛋白G2A κ型抗体，单克隆抗体APN 3FD9、3F10和10F3属于免疫球蛋白M型，经过加工的kappa轻链。这些单克隆抗体中的大多数显示AV值超过50%，表明它们靶向APN中的不同表位，而APN 5C51抗体识别抗原表位，如APN 3C48，5B31和6C56单克隆抗体识别的抗原表位。将扩增的APN 5B36 VHVL基因连接到pET28a阳性或pIRES2-zs Green载体中，分别构建重组表达质粒pET28a阳性RABS APN和pIRES2-zs Green 1 RABS APN。使用ELISA和IFA测定纯化和分析pET28a加重组抗体氨基肽酶NBL21和pIRES2-zs Green one重组抗体氨基肽酶NCHO细胞表达的抗体。然而，只有pIRES2-zs绿色一重组抗体氨基肽酶NCHO细胞上清液中表达的抗体才能识别APN蛋白。APN 5B36单克隆抗体与APN蛋白的结合比重组抗体更早达到平衡。使用蛋白A琼脂糖纯化上清液中的单克隆抗体比使用饱和硫酸氨沉淀更好。

Research

JoVE Journal

Immunology and Infection

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

单克隆抗体的反对消极行政 H。 capsulatum和其他真菌病原体

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

科研

免疫与感染

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

鼠单克隆抗体的产生杂交瘤技术

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

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

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

中和流感病毒单克隆抗体逃逸变种的产生

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 ...

Generation of Monoclonal Antibodies Against Natural Products

针对天然产物的单克隆抗体的产生

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...