Expression and Purification of the scFv from hybridoma cells secreting a monoclonal antibody against S PROTEIN of PEDV.
Identifieur interne : 001941 ( Main/Exploration ); précédent : 001940; suivant : 001942Expression and Purification of the scFv from hybridoma cells secreting a monoclonal antibody against S PROTEIN of PEDV.
Auteurs : Qinghe Zhu [République populaire de Chine] ; Donghua Guo ; Li Feng ; Dongbo SunSource :
- Monoclonal antibodies in immunodiagnosis and immunotherapy [ 2167-9436 ] ; 2013.
Descripteurs français
- KwdFr :
- Amorces ADN (génétique), Analyse de séquence d'ADN, Anticorps monoclonaux (immunologie), Anticorps à chaîne unique (génétique), Anticorps à chaîne unique (métabolisme), Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires (immunologie), Hybridomes (immunologie), Isopropyl-1-thio-bêta-D-galactopyranoside, Oligopeptides (immunologie), Protéines de l'enveloppe virale (immunologie), RT-PCR, Technique de Western, Virus de la diarrhée porcine épidémique (), Virus de la diarrhée porcine épidémique (immunologie).
- MESH :
- génétique : Amorces ADN, Anticorps à chaîne unique.
- immunologie : Anticorps monoclonaux, Glycoprotéines membranaires, Hybridomes, Oligopeptides, Protéines de l'enveloppe virale, Virus de la diarrhée porcine épidémique.
- métabolisme : Anticorps à chaîne unique.
- Analyse de séquence d'ADN, Glycoprotéine de spicule des coronavirus, Isopropyl-1-thio-bêta-D-galactopyranoside, RT-PCR, Technique de Western, Virus de la diarrhée porcine épidémique.
English descriptors
- KwdEn :
- Antibodies, Monoclonal (immunology), Blotting, Western, DNA Primers (genetics), Hybridomas (immunology), Isopropyl Thiogalactoside, Membrane Glycoproteins (immunology), Oligopeptides (immunology), Porcine epidemic diarrhea virus (chemistry), Porcine epidemic diarrhea virus (immunology), Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Single-Chain Antibodies (genetics), Single-Chain Antibodies (metabolism), Spike Glycoprotein, Coronavirus, Viral Envelope Proteins (immunology).
- MESH :
- chemical , genetics : DNA Primers, Single-Chain Antibodies.
- chemical , immunology : Antibodies, Monoclonal, Membrane Glycoproteins, Oligopeptides, Viral Envelope Proteins.
- chemistry : Porcine epidemic diarrhea virus.
- immunology : Hybridomas, Porcine epidemic diarrhea virus.
- chemical , metabolism : Single-Chain Antibodies.
- Blotting, Western, Isopropyl Thiogalactoside, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Spike Glycoprotein, Coronavirus.
Abstract
The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the recombinant scFv/6E6 protein was purified from the inclusion body form by the gel-cutting measure followed by electroelution and dialysis. The recombinant scFv/6E6 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule.
DOI: 10.1089/mab.2012.0089
PubMed: 23600505
Affiliations:
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Le document en format XML
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sort="Feng, Li" uniqKey="Feng L" first="Li" last="Feng">Li Feng</name></author><author><name sortKey="Sun, Dongbo" sort="Sun, Dongbo" uniqKey="Sun D" first="Dongbo" last="Sun">Dongbo Sun</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2013">2013</date><idno type="RBID">pubmed:23600505</idno><idno type="pmid">23600505</idno><idno type="doi">10.1089/mab.2012.0089</idno><idno type="wicri:Area/PubMed/Corpus">001214</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">001214</idno><idno type="wicri:Area/PubMed/Curation">001214</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">001214</idno><idno type="wicri:Area/PubMed/Checkpoint">001182</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">001182</idno><idno type="wicri:Area/Ncbi/Merge">002658</idno><idno type="wicri:Area/Ncbi/Curation">002658</idno><idno 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Daqing</wicri:regionArea><wicri:noRegion>Daqing</wicri:noRegion></affiliation></author><author><name sortKey="Guo, Donghua" sort="Guo, Donghua" uniqKey="Guo D" first="Donghua" last="Guo">Donghua Guo</name></author><author><name sortKey="Feng, Li" sort="Feng, Li" uniqKey="Feng L" first="Li" last="Feng">Li Feng</name></author><author><name sortKey="Sun, Dongbo" sort="Sun, Dongbo" uniqKey="Sun D" first="Dongbo" last="Sun">Dongbo Sun</name></author></analytic><series><title level="j">Monoclonal antibodies in immunodiagnosis and immunotherapy</title><idno type="eISSN">2167-9436</idno><imprint><date when="2013" type="published">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Antibodies, Monoclonal (immunology)</term><term>Blotting, Western</term><term>DNA Primers (genetics)</term><term>Hybridomas (immunology)</term><term>Isopropyl Thiogalactoside</term><term>Membrane Glycoproteins (immunology)</term><term>Oligopeptides (immunology)</term><term>Porcine epidemic diarrhea virus (chemistry)</term><term>Porcine epidemic diarrhea virus (immunology)</term><term>Reverse Transcriptase Polymerase Chain Reaction</term><term>Sequence Analysis, DNA</term><term>Single-Chain Antibodies (genetics)</term><term>Single-Chain Antibodies (metabolism)</term><term>Spike Glycoprotein, Coronavirus</term><term>Viral Envelope Proteins (immunology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Amorces ADN (génétique)</term><term>Analyse de séquence d'ADN</term><term>Anticorps monoclonaux (immunologie)</term><term>Anticorps à chaîne unique (génétique)</term><term>Anticorps à chaîne unique (métabolisme)</term><term>Glycoprotéine de spicule des coronavirus</term><term>Glycoprotéines membranaires (immunologie)</term><term>Hybridomes (immunologie)</term><term>Isopropyl-1-thio-bêta-D-galactopyranoside</term><term>Oligopeptides (immunologie)</term><term>Protéines de l'enveloppe virale (immunologie)</term><term>RT-PCR</term><term>Technique de Western</term><term>Virus de la diarrhée porcine épidémique ()</term><term>Virus de la diarrhée porcine épidémique (immunologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA Primers</term><term>Single-Chain Antibodies</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Antibodies, Monoclonal</term><term>Membrane Glycoproteins</term><term>Oligopeptides</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Porcine epidemic diarrhea virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Amorces ADN</term><term>Anticorps à chaîne unique</term></keywords><keywords scheme="MESH" qualifier="immunologie" xml:lang="fr"><term>Anticorps monoclonaux</term><term>Glycoprotéines membranaires</term><term>Hybridomes</term><term>Oligopeptides</term><term>Protéines de l'enveloppe virale</term><term>Virus de la diarrhée porcine épidémique</term></keywords><keywords scheme="MESH" qualifier="immunology" xml:lang="en"><term>Hybridomas</term><term>Porcine epidemic diarrhea virus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Single-Chain Antibodies</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Anticorps à chaîne unique</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Blotting, Western</term><term>Isopropyl Thiogalactoside</term><term>Reverse Transcriptase Polymerase Chain Reaction</term><term>Sequence Analysis, DNA</term><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Analyse de séquence d'ADN</term><term>Glycoprotéine de spicule des coronavirus</term><term>Isopropyl-1-thio-bêta-D-galactopyranoside</term><term>RT-PCR</term><term>Technique de Western</term><term>Virus de la diarrhée porcine épidémique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the recombinant scFv/6E6 protein was purified from the inclusion body form by the gel-cutting measure followed by electroelution and dialysis. The recombinant scFv/6E6 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><noCountry><name sortKey="Feng, Li" sort="Feng, Li" uniqKey="Feng L" first="Li" last="Feng">Li Feng</name><name sortKey="Guo, Donghua" sort="Guo, Donghua" uniqKey="Guo D" first="Donghua" last="Guo">Donghua Guo</name><name sortKey="Sun, Dongbo" sort="Sun, Dongbo" uniqKey="Sun D" first="Dongbo" last="Sun">Dongbo Sun</name></noCountry><country name="République populaire de Chine"><noRegion><name sortKey="Zhu, Qinghe" sort="Zhu, Qinghe" uniqKey="Zhu Q" first="Qinghe" last="Zhu">Qinghe Zhu</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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