Generating stable chinese hamster ovary cell clones to produce a truncated SARS‐CoV spike protein for vaccine development
Identifieur interne : 001951 ( Main/Merge ); précédent : 001950; suivant : 001952Generating stable chinese hamster ovary cell clones to produce a truncated SARS‐CoV spike protein for vaccine development
Auteurs : Shih-Chang Lin [Taïwan] ; Chih-Hsiang Leng [Taïwan] ; Suh-Chin Wu [Taïwan]Source :
- Biotechnology Progress [ 8756-7938 ] ; 2010-11.
English descriptors
- Teeft :
- Biotechnol, Biotechnol bioeng, Cell clones, Cells transfected, Chinese hamster ovary cells, Clone, Coronavirus, Dhfr, Dhfr expression, Dhfr gene, Dhfr protein, Gene, Glycoform, Isid, Isid clones, Isid vector, Isiz, Isiz clones, Isiz vector, Mammalian cells, National tsing, Neutralizing, Neutralizing antibodies, Proc natl acad, Prog, Promoter, Protective immunity, Recombinant, Respiratory syndrome coronavirus, Spike, Spike protein, Stable cell clones, Stable clones, Str2, Str2 expression, Str2 glycoform, Str2 protein, Subunit, Subunit vaccine, Transfected, Transfected cells, Vaccine, Western blot analysis.
Abstract
The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS‐CoV) is important for vaccine development. STR2 (an 88 kDa truncated SARS‐CoV TW1 S protein carrying the S fragments S‐74‐253, S‐294‐739, and S‐1129‐1255) is capable of expressing a major form of glycoprotein as endo H‐sensitive (∼115 kDa) in CHO cells. To establish stable expressing cell clones, we transfected CHO/dhFr‐cells with the amplifiable vectors ISID (IRES‐driven dhfr) and ISIZ (SV40‐driven dhfr) to select stepwise MTX, and observed enhanced ∼115 kDa glycoform generation through gene amplification. Following stepwise MTX selection, we compared gene amplification levels between two vectors in engineered CHO cell chromosomes. These results confirm that the IRES‐driven dhfr promoter generates greater gene amplification, which in turn enhances STR2 expression. Our results indicate that the ∼115 kDa glycoform of STR2 protein was capable of increasing after gene amplification. The STR2 glycoform did not change between suspension and serum‐free cultures, suggesting that the stable and amplified cell clones analyzed in this study have potential for producing homologous STR2 on a large scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010
Url:
- https://api.istex.fr/ark:/67375/WNG-5F6M70PD-3/fulltext.pdf
- http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161902
DOI: 10.1002/btpr.480
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wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Vaccine Research and Development Center, National Health Research Institutes, Zhunan 350</wicri:regionArea><wicri:noRegion>Zhunan 350</wicri:noRegion></affiliation></author><author><name sortKey="Wu, Suh Hin" sort="Wu, Suh Hin" uniqKey="Wu S" first="Suh-Chin" last="Wu">Suh-Chin Wu</name><affiliation></affiliation><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Vaccine Research and Development Center, National Health Research Institutes, Zhunan 350</wicri:regionArea><wicri:noRegion>Zhunan 350</wicri:noRegion></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Taïwan</country></affiliation><affiliation wicri:level="1"><country xml:lang="fr" wicri:curation="lc">Taïwan</country><wicri:regionArea>Correspondence address: Institute of Biotechnology, Dept. of Life Science, National Tsing Hua University, Hsinchu 30013</wicri:regionArea><wicri:noRegion>Hsinchu 30013</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biotechnology Progress</title><title level="j" type="alt">BIOTECHNOLOGY PROGRESS</title><idno type="ISSN">8756-7938</idno><idno type="eISSN">1520-6033</idno><imprint><biblScope unit="vol">26</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="1733">1733</biblScope><biblScope unit="page" to="1740">1740</biblScope><biblScope unit="page-count">8</biblScope><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><pubPlace>Hoboken</pubPlace><date type="published" when="2010-11">2010-11</date></imprint><idno type="ISSN">8756-7938</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">8756-7938</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Biotechnol</term><term>Biotechnol bioeng</term><term>Cell clones</term><term>Cells transfected</term><term>Chinese hamster ovary cells</term><term>Clone</term><term>Coronavirus</term><term>Dhfr</term><term>Dhfr expression</term><term>Dhfr gene</term><term>Dhfr protein</term><term>Gene</term><term>Glycoform</term><term>Isid</term><term>Isid clones</term><term>Isid vector</term><term>Isiz</term><term>Isiz clones</term><term>Isiz vector</term><term>Mammalian cells</term><term>National tsing</term><term>Neutralizing</term><term>Neutralizing antibodies</term><term>Proc natl acad</term><term>Prog</term><term>Promoter</term><term>Protective immunity</term><term>Recombinant</term><term>Respiratory syndrome coronavirus</term><term>Spike</term><term>Spike protein</term><term>Stable cell clones</term><term>Stable clones</term><term>Str2</term><term>Str2 expression</term><term>Str2 glycoform</term><term>Str2 protein</term><term>Subunit</term><term>Subunit vaccine</term><term>Transfected</term><term>Transfected cells</term><term>Vaccine</term><term>Western blot analysis</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS‐CoV) is important for vaccine development. STR2 (an 88 kDa truncated SARS‐CoV TW1 S protein carrying the S fragments S‐74‐253, S‐294‐739, and S‐1129‐1255) is capable of expressing a major form of glycoprotein as endo H‐sensitive (∼115 kDa) in CHO cells. To establish stable expressing cell clones, we transfected CHO/dhFr‐cells with the amplifiable vectors ISID (IRES‐driven dhfr) and ISIZ (SV40‐driven dhfr) to select stepwise MTX, and observed enhanced ∼115 kDa glycoform generation through gene amplification. Following stepwise MTX selection, we compared gene amplification levels between two vectors in engineered CHO cell chromosomes. These results confirm that the IRES‐driven dhfr promoter generates greater gene amplification, which in turn enhances STR2 expression. Our results indicate that the ∼115 kDa glycoform of STR2 protein was capable of increasing after gene amplification. The STR2 glycoform did not change between suspension and serum‐free cultures, suggesting that the stable and amplified cell clones analyzed in this study have potential for producing homologous STR2 on a large scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010</div></front></TEI><double doi="10.1002/btpr.480"><ISTEX><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Generating stable chinese hamster ovary cell clones to produce a truncated SARS‐CoV spike protein for vaccine development</title><author><name sortKey="Lin, Shih Hang" sort="Lin, Shih Hang" uniqKey="Lin S" first="Shih-Chang" last="Lin">Shih-Chang Lin</name></author><author><name sortKey="Leng, Chih Siang" sort="Leng, Chih Siang" uniqKey="Leng C" first="Chih-Hsiang" last="Leng">Chih-Hsiang Leng</name></author><author><name sortKey="Wu, Suh Hin" sort="Wu, Suh Hin" uniqKey="Wu S" first="Suh-Chin" last="Wu">Suh-Chin Wu</name></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:9CD39EE81911099DB22873A60E5DCA5EEFDCD023</idno><date when="2010" year="2010">2010</date><idno type="doi">10.1002/btpr.480</idno><idno 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University, Hsinchu 30013</wicri:regionArea><wicri:noRegion>Hsinchu 30013</wicri:noRegion></affiliation></author><author><name sortKey="Leng, Chih Siang" sort="Leng, Chih Siang" uniqKey="Leng C" first="Chih-Hsiang" last="Leng">Chih-Hsiang Leng</name><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Vaccine Research and Development Center, National Health Research Institutes, Zhunan 350</wicri:regionArea><wicri:noRegion>Zhunan 350</wicri:noRegion></affiliation></author><author><name sortKey="Wu, Suh Hin" sort="Wu, Suh Hin" uniqKey="Wu S" first="Suh-Chin" last="Wu">Suh-Chin Wu</name><affiliation></affiliation><affiliation wicri:level="1"><country xml:lang="fr">Taïwan</country><wicri:regionArea>Vaccine Research and Development Center, National Health Research Institutes, Zhunan 350</wicri:regionArea><wicri:noRegion>Zhunan 350</wicri:noRegion></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Taïwan</country></affiliation><affiliation wicri:level="1"><country xml:lang="fr" wicri:curation="lc">Taïwan</country><wicri:regionArea>Correspondence address: Institute of Biotechnology, Dept. of Life Science, National Tsing Hua University, Hsinchu 30013</wicri:regionArea><wicri:noRegion>Hsinchu 30013</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biotechnology Progress</title><title level="j" type="alt">BIOTECHNOLOGY PROGRESS</title><idno type="ISSN">8756-7938</idno><idno type="eISSN">1520-6033</idno><imprint><biblScope unit="vol">26</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="1733">1733</biblScope><biblScope unit="page" to="1740">1740</biblScope><biblScope unit="page-count">8</biblScope><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><pubPlace>Hoboken</pubPlace><date type="published" when="2010-11">2010-11</date></imprint><idno type="ISSN">8756-7938</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">8756-7938</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Biotechnol</term><term>Biotechnol bioeng</term><term>Cell clones</term><term>Cells transfected</term><term>Chinese hamster ovary cells</term><term>Clone</term><term>Coronavirus</term><term>Dhfr</term><term>Dhfr expression</term><term>Dhfr gene</term><term>Dhfr protein</term><term>Gene</term><term>Glycoform</term><term>Isid</term><term>Isid clones</term><term>Isid vector</term><term>Isiz</term><term>Isiz clones</term><term>Isiz vector</term><term>Mammalian cells</term><term>National tsing</term><term>Neutralizing</term><term>Neutralizing antibodies</term><term>Proc natl acad</term><term>Prog</term><term>Promoter</term><term>Protective immunity</term><term>Recombinant</term><term>Respiratory syndrome coronavirus</term><term>Spike</term><term>Spike protein</term><term>Stable cell clones</term><term>Stable clones</term><term>Str2</term><term>Str2 expression</term><term>Str2 glycoform</term><term>Str2 protein</term><term>Subunit</term><term>Subunit vaccine</term><term>Transfected</term><term>Transfected cells</term><term>Vaccine</term><term>Western blot analysis</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS‐CoV) is important for vaccine development. STR2 (an 88 kDa truncated SARS‐CoV TW1 S protein carrying the S fragments S‐74‐253, S‐294‐739, and S‐1129‐1255) is capable of expressing a major form of glycoprotein as endo H‐sensitive (∼115 kDa) in CHO cells. To establish stable expressing cell clones, we transfected CHO/dhFr‐cells with the amplifiable vectors ISID (IRES‐driven dhfr) and ISIZ (SV40‐driven dhfr) to select stepwise MTX, and observed enhanced ∼115 kDa glycoform generation through gene amplification. Following stepwise MTX selection, we compared gene amplification levels between two vectors in engineered CHO cell chromosomes. These results confirm that the IRES‐driven dhfr promoter generates greater gene amplification, which in turn enhances STR2 expression. Our results indicate that the ∼115 kDa glycoform of STR2 protein was capable of increasing after gene amplification. The STR2 glycoform did not change between suspension and serum‐free cultures, suggesting that the stable and amplified cell clones analyzed in this study have potential for producing homologous STR2 on a large scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010</div></front></TEI></ISTEX><PMC><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Generating stable chinese hamster ovary cell clones to produce a truncated SARS‐CoV spike protein for vaccine development</title><author><name sortKey="Lin, Shih Hang" sort="Lin, Shih Hang" uniqKey="Lin S" first="Shih-Chang" last="Lin">Shih-Chang Lin</name><affiliation><nlm:aff id="af1"></nlm:aff></affiliation></author><author><name sortKey="Leng, Chih Siang" sort="Leng, Chih Siang" uniqKey="Leng C" first="Chih-Hsiang" last="Leng">Chih-Hsiang Leng</name><affiliation><nlm:aff id="af2"></nlm:aff></affiliation></author><author><name sortKey="Wu, Suh Hin" sort="Wu, Suh Hin" uniqKey="Wu S" first="Suh-Chin" last="Wu">Suh-Chin Wu</name><affiliation><nlm:aff id="af3"></nlm:aff></affiliation><affiliation><nlm:aff id="af4"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">20809484</idno><idno type="pmc">7161902</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161902</idno><idno type="RBID">PMC:7161902</idno><idno type="doi">10.1002/btpr.480</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">000615</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000615</idno><idno type="wicri:Area/Pmc/Curation">000615</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000615</idno><idno type="wicri:Area/Pmc/Checkpoint">000884</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000884</idno><idno type="wicri:Area/Ncbi/Merge">000400</idno><idno type="wicri:Area/Ncbi/Curation">000400</idno><idno type="wicri:Area/Ncbi/Checkpoint">000400</idno><idno type="wicri:doubleKey">8756-7938:2010:Lin S:generating:stable:chinese</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Generating stable chinese hamster ovary cell clones to produce a truncated SARS‐CoV spike protein for vaccine development</title><author><name sortKey="Lin, Shih Hang" sort="Lin, Shih Hang" uniqKey="Lin S" first="Shih-Chang" last="Lin">Shih-Chang Lin</name><affiliation><nlm:aff id="af1"></nlm:aff></affiliation></author><author><name sortKey="Leng, Chih Siang" sort="Leng, Chih Siang" uniqKey="Leng C" first="Chih-Hsiang" last="Leng">Chih-Hsiang Leng</name><affiliation><nlm:aff id="af2"></nlm:aff></affiliation></author><author><name sortKey="Wu, Suh Hin" sort="Wu, Suh Hin" uniqKey="Wu S" first="Suh-Chin" last="Wu">Suh-Chin Wu</name><affiliation><nlm:aff id="af3"></nlm:aff></affiliation><affiliation><nlm:aff id="af4"></nlm:aff></affiliation></author></analytic><series><title level="j">Biotechnology Progress</title><idno type="ISSN">8756-7938</idno><idno type="eISSN">1520-6033</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Abstract</title><p>The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS‐CoV) is important for vaccine development. S<sub>TR2</sub> (an 88 kDa truncated SARS‐CoV TW1 S protein carrying the S fragments S‐74‐253, S‐294‐739, and S‐1129‐1255) is capable of expressing a major form of glycoprotein as endo H‐sensitive (∼115 kDa) in CHO cells. To establish stable expressing cell clones, we transfected CHO/dhFr‐cells with the amplifiable vectors ISID (IRES‐driven dhfr) and ISIZ (SV40‐driven dhfr) to select stepwise MTX, and observed enhanced ∼115 kDa glycoform generation through gene amplification. Following stepwise MTX selection, we compared gene amplification levels between two vectors in engineered CHO cell chromosomes. These results confirm that the IRES‐driven <italic>dhfr</italic> promoter generates greater gene amplification, which in turn enhances S<sub>TR2</sub> expression. Our results indicate that the ∼115 kDa glycoform of S<sub>TR2</sub> protein was capable of increasing after gene amplification. The S<sub>TR2</sub> glycoform did not change between suspension and serum‐free cultures, suggesting that the stable and amplified cell clones analyzed in this study have potential for producing homologous S<sub>TR2</sub> on a large scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI></PMC></double></record>Pour manipuler ce document sous Unix (Dilib)
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