Name: peptide scanning-assisted identification of a monoclonal antibody-recognized linear b-cell epitope
Uploaded: 2017-03-24T14:00:00
Description: Watch this Scientific Journal Video about Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope at JoVE.com

The authors thank Miss Ching-Chun Lin and Miss Diana Lin of the Core Facility of the Institute of Cellular and Organismic Biology (ICOB) of Academia Sinica for offering their expertise on peptide synthesis and DNA sequencing, respectively. This study was supported by Academia Sinica.

Monoklonal antikor hazırlanması 1. <ol><li> Kültür,% 5 CO2 ilave ile 37 ° C &#39;de 175T şişelerinde serum içermeyen ortam içinde RG-M56 tek klonlu fare hibridoma hücreleri, 2. orta renk inkübasyon beş gün sonra sarı döndüğünde süpernatant toplayın. Not: hibridoma hücreleri cenin sığır serumu antikor kirlenmesini önlemek için, serum barındırmayan ortam içinde kültürlenmiştir. </li><li> 4 ° C&#39;de 30 dakika boyunca 4500 x g&#39;de santrifüj s...

<ol>
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Bu protokol, mAb tanınan lineer epitopunu belirlemek için hızlı ve basit bir teknik sunmaktadır. dikkate peptit sentezi ve sentez peptidlerin üretim verimliliği maliyeti alarak, virüs kaplama proteininin antijenik bölgenin peptit tarama analizinden önce seri kesik rekombinant proteinleri ifade azaltılmıştır. 10 ila 50 arasındaki moleküler ağırlıklara sahip bir araya getiren proteinler kDa kolaylıkla bu sistem ile ifade edilebilir Bu nedenle, güvenli ve etkili bir E.coli pET ifade sistemi, b...

Bağışıklık sisteminde, V, D ve J segmentlerinin rekombinasyon antikorları patojenik enfeksiyon ana korumak için çeşitli antijenlere bağlanması için tamamlayıcılığı belirleyici bölgelerinde (CDR&#39;ler) muazzam varyasyonlarını oluşturmak için sağlar. antijenlere karşı antikorların nötralize savunma antikorlarının CDR ve antijenlerin epitoplarına arasındaki mekansal tamamlayıcılık bağlıdır. Bu nedenle, bu moleküler etkileşim bir anlayış profilaktik aşı tasarımı ve terapötik peptid ilaç geliştirme yardımcı olacaktır. Bununla birlikte, bu nötrleştirme etkileşimi, tek bir antijen birden fazla antijenik alan ile ve dolayısıyla epitop belirleme süreci daha karmaşık hale antikor çoklu CDR&#39;ler, hem de etkilenebilir. Neyse ki, miyelom hücreleri ile, bireysel antikor üreten hücrelerin sigortalar hibridom teknolojisinin gelişmesi, sn hücrelerin sürekli bölünmesi toplu sağlarBir monoklonal antikor (mAb) olarak bilinen retenin belirli bir antikor, 1. Hibridoma hücreleri, spesifik antijenin tek bir antijenik alana bağlanabilir, bu, saf, yüksek afiniteli mAbs üretir. Kurulan antijen-antikor, peptit taraması dahil olmak üzere çeşitli yaklaşımlar arasında bir ilişki ile, karşılık gelen mAb kullanan bir antijenin epitopunun belirlenmesi için kullanılabilir. sentetik peptid teknolojisindeki son gelişmeler peptid tarama tekniği daha erişilebilir ve gerçekleştirmek için daha uygun hale getirmiştir. Kısaca, üst üste binen sentetik peptidlere bir dizi, bir hedef bir antijen dizisine göre üretilir mAb hibridizasyona katı destekli zara ilişkilidir. Peptid tarama bölgesinin antikor bağlanmasını eşlemek için kolay bir yol sunar, ancak, aynı zamanda epitop peptit her AA tortu antikorun CDR arasındaki bağlanma etkileşimini değerlendirmek için tortu, tarama veya ikame arası amino asit (aa) mutagenez kolaylaştırmaktadır. 2, 3, 4 ile san bir orfoz sinir nekroz virüsü (YGNNV) kaplama proteininin lineer epitopa etkin tanımlanması için bir protokol açıklamaktadır. Protokol mAb, inşaat ve seri kesik rekombinant proteinlerin ekspresyonu sentetik üstüste binen peptit tasarım, nokta-benek hibridizasyonu, alanin tarama ve ikame mutagenezi bulunmaktadır. Peptit sentezi yüksek maliyeti göz önüne alındığında, seri arzu edilen bir hedef proteini rekombinant proteinleri kesilmesi aşamasının tadil edilmiş, ve sentetik peptid dizisi nokta-blot analizi gerçekleştirilmiştir önce antijenik bölge yaklaşık 100 ila 200 aa artıkları kadar daraltılmıştır.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hybrid-SFM medium</td>
			<td>Gibco</td>
			<td>12045-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeccos&#39;s Phophate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu DNA Polymerase&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>EP0502&#160;</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Roche</td>
			<td>10799009001</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>NdeI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>XhoI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>pET-20b(+) vector</td>
			<td>Novagen, Merck Millipore</td>
			<td>69739</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli DH-5&#945; competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH617</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli BL-21(DE3) competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH217</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Amresco</td>
			<td>0339-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth&#160;</td>
			<td>Invitrongen</td>
			<td>12780-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropylthio-&#946;-D-thiogalactoside (IPTG)</td>
			<td>MDBio, Inc.</td>
			<td>101-367-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T.Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Amresco</td>
			<td>0167-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Affymetrix, USB</td>
			<td>75825</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Amresco</td>
			<td>0241-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Amresco</td>
			<td>0105-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Amresco</td>
			<td>0854-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>PerkinElmer</td>
			<td>NEL937001PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG, Fc fragment antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-055-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P (Polyvinylidene fluoride, PVDF)</td>
			<td>Merck Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Agarose Fast Flow</td>
			<td>Merck Millipore</td>
			<td>16-266</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP BioSpectrum 600 Image System</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>VisionWorks LS Analysis Software Ver 6.8</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>MyCycler thermal cycler</td>
			<td>BioRad</td>
			<td>1709713</td>
			<td></td>
		</tr>
	</tbody>
</table>

peptide scanning-assisted identification of a monoclonal antibody-recognized linear b-cell epitope

bağışıklık sisteminin bir antijenik epitopa tanımlanması aşılar ve peptid ilaçların geliştirilmesi kolaylaştırabilir nötralize edici antikorların bir koruyucu mekanizma anlaşılabilmiştir. Peptid tarama delikanlı monoklonal bir antikor (mAb) tarafından tanınan lineer epitop harita basit ve etkili bir yöntemdir. Burada, yazarlar nötrleştirici mAb kullanılarak sinir nekroz virüsü kaplama proteininin antigenik tanınması için seri kesik rekombinant proteinleri, sentetik peptid tasarımı ve nokta-benek hibridizasyonu ile ilgili bir epitop belirlenmesi yöntemi sunulmaktadır. Bu teknik, bir poliviniliden florür (PVDF) membran üzerinde sentetik peptidler ve mAb&#39;lerin nokta benek hibridizasyonu dayanır. RG-M56 mAb tarafından tanınan bir viral kat proteininin en az antijenik bölge 6-mer peptit epitopu üzerine peptit haritalama kesilmiş adım adım daraltıldığı edilebilir. Buna ek olarak, alanin tarama mutajenezi ve tortu, altikamesi veya epitopu oluşturan her bir amino asit tortusu bağlanması önem karakterize etmek için gerçekleştirilebilir. epitop sitesini kuşatan kalıntıları peptid konformasyon düzenlenmesinde kritik roller oynamaya bulundu. tanımlanan epitop peptit bir X-ışını kırılım çalışmasının ve fonksiyonel rekabet veya tedavi için epitopunun peptid-antikor komplekslerinin kristalleri meydana getirmek üzere kullanılabilir.

Bu deneyin amacı, mAb kullanılarak nokta blotlama yoluyla bir epitopu belirlemek için yapıldı. Hızlı ve verimli bir şekilde mAb tarafından algılanan antijenik bölge daraltmak için, C-terminali bir 6xHis füzyon etiketidir tam boyunda ve seri olarak kesildi YGNNV rekombinant kaplama proteinleri bir E. coli pET ifade sistemi 10 (Şekil 1 A) ile ilgili olarak ifade edildi. Elde edilen rekombinant proteinleri nokta benek hibridiza...

Watch this Scientific Journal Video about Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope at JoVE.com

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Peptid, bir monoklonal antikor tarafından tanınan lineer B-hücresi epitopunun belirlenmesi: Tarama destekli

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

The overall goal of this protocol is to identify a monoclonal antibody-recognized linear B-cell epitope using serially truncated recombinant proteins and peptide scanning by dot-blot hybridization. This method can help answer key questions concerning the molecule interaction between antigen and the antibody. The main advantages of this technique are its simplicity and efficiency.The identifying epitope can then be used in fuller investigations such as in therapeutical and diagnostic applications. Demonstrating the procedure will be Chien-Wen Chen, a post doctorate from my laboratory. To begin, grow RGM56 mouse monoclonal hybridoma cells in a hybrid serum free medium.Maintain cell cultures in 175 T flasks at 37 degrees Celsius 5%carbon dioxide. After five days of incubation, collect the culture medium after it has turned yellow and centrifuge the supernatant at 4, 500 times G for 30 minutes at four degrees Celsius. Then, discard the pelleted cell debris to obtain the antibody in solution.Next, add two milliliters of 50%protein G agro slurry to a five milliliter column and equilibrate the resin with 10 milliliters of ice cold PBS, equaling 10 volumes of preloaded resin. Load 200 milliliters of the previously prepared antibody solution onto the equilibrated column and discard the fraction that passes through the column. Using 10 milliliters of ice cold PBS, wash the column twice.Finally, to loot the antibody from the resin, add 10 milliliters of millimolar glycerin solution at pH 2.7 to the column. Collect 900 microliter fractions in microcentrifuge tubes previously filled with 100 microliters of 10X neutralization buffer. Store the purified antibody in 50%glycerol solution supplemented with sodium azide at minus 20 degrees Celsius.Prepare the constructs and transform E.coli BL21 cells as described in the text protocol. Then, transfer a single colony into a 15 milliliter tube containing three milliliters of LB broth supplemented with 100 micrograms per milliliter ampicillin and loosely cap the tube. Incubate the culture at 37 degrees Celsius with 150 RPM shaking.Monitor the optical density of the culture at 600 nanometers. When it reaches about 0.6, cool the culture to 25 degrees Celsius. Then, add IPTG at a final concentration of 0.4 millimolar to induce the expression of the recombinant protein.Incubate the culture for another four hours at 25 degrees Celsius with 200 RPM shaking. After incubation, transfer one milliliter of the bacterial culture into a microcentrifuge tube. Centrifuge the cells at 12, 000 times G for one minute and discard the supernatant.Pipetting up and down with a micropipette, resuspend the cell pellet in 100 microliters of denaturation buffer. And mix the resulting suspension by vigorous vortexing to obtain a ready to use recombinant protein sample. To proceed with dot-blot hybridization, dissolve each synthetic peptide in DMSO to obtain 10 milligram per milliliter solutions.Then, activate the PVDF membrane in methanol for two minutes and equilibrate it with modified Towbin buffer for another two minutes. Rinse a piece of chromatography paper with modified Towbin buffer and lay the equilibrated PVDF membrane on it. Let the membrane sit until the buffer disappears from its surface.Using a 10 microliter tip, add two microliters of each peptide or recombinant protein sample onto the membrane and let it air dry for 10 minutes. Spotting a symbol onto the membrane is the most critical step in this protocol. Loading a precise amount of a sample onto a small area of the PVDF membrane needs careful attention.Next, block the membrane in TBST buffer supplemented with 5%nonfat milk for 30 minutes at room temperature with gentle shaking. Dilute the RGM56 monoclonal antibody in TBST buffer supplemented with 5%nonfat milk. And add the antibody solution to the membrane.Incubate the membrane at 37 degrees Celsius for one hour with gentle shaking. After incubation, remove the antibody solution. And wash the membrane twice in TBST buffer for five minutes with gentle shaking.Then, add to the membrane the solution of the secondary antibody diluted in TBST buffer with 5%nonfat milk. Incubate the membrane for one hour at 37 degrees Celsius with gentle shaking. Upon incubation, discard the antibody solution and wash the membrane in TBST buffer twice for five minutes with gentle shaking.Then, develop the membrane with the substrate solution at room temperature for 15 minutes in the dark. When the signal appears, stop the reaction by washing the membrane with doubly distilled water. After air drying the membrane, capture the dot-blot image using an imaging system.Finally, using image analysis software measure the intensity of each dot-blot. Presented here is the full length nervous necrosis virus code protein comprising amino acids one through 338 along with its serially truncated variants. To confirm the expression of the proteins in the transformed bacterial cells, dot-blot analysis using anti-6XHis antibody was performed showing the presence of all the proteins under investigation.Then, the recombinant proteins were analyzed by dot-blot hybridization in which the RG-M56 monoclonal antibody was used. The analysis revealed that the protein epitope recognized by the antibody is found in peptide 195 to 338. Finally, an epitope consisting of 20 or eight amino acid residues was identified by peptide scanning.Alanine scanning showed that substitutions of valine residues at positions 197 and 199 as well as a cystine at 201 abolished the epitope's binding capability and dot-blot hybridization. Similarly, the decreased binding affinity was observed in binding intensity analysis confirming that the identified residues are essential for the epitope's binding to the RGM56 monoclonal antibody. This peptide identification technique can be used to develop therapeutical peptide drugs or peptide medicines.

Research

JoVE Journal

Immunology and Infection

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Peptid: Epitop-spesifik T-hücrelerinin MHC tetramer bazlı Zenginleştirme

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

İmmünoloji ve Enfeksiyon

Peptide-based Identification of Functional Motifs and their Binding Partners

Fonksiyonel Motifler ve Bağlama Ortaklar peptid bazlı Kimlik

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Kordon Kanı gelen Prekürsör B-hücreli altkümeleri İzolasyonu

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Hibridoma teknolojisi ile murin monoklonal antikorlar üretilmesi

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

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Maymun bağışıklık eksikliği virüsüne özgü CD8 analizi<sup&gt; +</sup&gt; Peptid MHC-I tetramer boyama ile Rhesus macaque maymunlarının T-hücrelerinin

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Grip virüsü monoklonal antikorları nötralize kaçış türevleri üretimi

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 ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

Teşhis edilmesi için kullanılan ilişkili Viral ve hücresel proteinler Viral DNA'ın arıtma

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 epitopları bir protein eşlemek için çakışan Peptide Kütüphane

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

Zika Virus Specific Diagnostic Epitope Discovery

Zika virüs belirli tanılama Epitope bulma

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...