Selection of scFvs specific for HBV DNA polymerase using ribosome display
We applied a ribosome display technique to a mouse scFv library to select single chain variable fragments (scFvs) specific for the terminal protein (TP) of hepatitis B virus (HBV) DNA polymerase. Synthetic TP-peptide was used as an antigen to obtain scFvs that recognize specific epitopes within the...
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| Veröffentlicht in: | Journal of immunological methods 2004, Vol.284 (1), p.147-157 |
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| container_title | Journal of immunological methods |
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| creator | Lee, Myung-Shin Kwon, Myung-Hee Kim, Kyongmin Hwang Shin, Ho-Joon Park, Sun Kim, Hyung-Il |
| description | We applied a ribosome display technique to a mouse scFv library to select single chain variable fragments (scFvs) specific for the terminal protein (TP) of hepatitis B virus (HBV) DNA polymerase. Synthetic TP-peptide was used as an antigen to obtain scFvs that recognize specific epitopes within the TP domain, the priming site of HBV DNA polymerase. The scFv DNA library was transcribed in vitro to mRNA for ribosome display. scFv–ribosome–mRNA complexes were produced using a rabbit reticulocyte lysate system, and were panned against TP-peptide under appropriate conditions. After each panning, putative scFv-encoding genes were recovered by RT-PCR, and the analysis showed that scFv-ribosome-mRNA complexes were specifically selected by the TP-peptide. We used a radioimmunoassay to show that the TP-peptide-specific scFv pools were enriched through the selection process. Selected scFvs showed binding activity for both the TP-peptide and the HBV DNA polymerase protein in an ELISA. Sequence analysis showed that each TP-specific scFv had a different sequence, and that random mutagenesis was mediated by ribosome display. |
| doi_str_mv | 10.1016/j.jim.2003.10.009 |
| format | Article |
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Synthetic TP-peptide was used as an antigen to obtain scFvs that recognize specific epitopes within the TP domain, the priming site of HBV DNA polymerase. The scFv DNA library was transcribed in vitro to mRNA for ribosome display. scFv–ribosome–mRNA complexes were produced using a rabbit reticulocyte lysate system, and were panned against TP-peptide under appropriate conditions. After each panning, putative scFv-encoding genes were recovered by RT-PCR, and the analysis showed that scFv-ribosome-mRNA complexes were specifically selected by the TP-peptide. We used a radioimmunoassay to show that the TP-peptide-specific scFv pools were enriched through the selection process. Selected scFvs showed binding activity for both the TP-peptide and the HBV DNA polymerase protein in an ELISA. Sequence analysis showed that each TP-specific scFv had a different sequence, and that random mutagenesis was mediated by ribosome display.</description><identifier>ISSN: 0022-1759</identifier><identifier>EISSN: 1872-7905</identifier><identifier>DOI: 10.1016/j.jim.2003.10.009</identifier><identifier>PMID: 14736425</identifier><identifier>CODEN: JIMMBG</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Amino Acid Sequence ; Animals ; Antibodies, Viral - blood ; Antibody Specificity ; Base Sequence ; Biological and medical sciences ; Chromatography, Affinity ; DNA-Directed DNA Polymerase - genetics ; DNA-Directed DNA Polymerase - immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Fundamental and applied biological sciences. Psychology ; Fundamental immunology ; HBV DNA polymerase ; Hepatitis B virus ; Hepatitis B virus - enzymology ; Hepatitis B virus - genetics ; Immunoglobulin Variable Region - genetics ; Immunoglobulin Variable Region - immunology ; Mice ; Mice, Inbred BALB C ; Microbiology ; Molecular immunology ; Molecular Sequence Data ; Protein Biosynthesis - immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Ribosome display ; Ribosomes - immunology ; RNA, Messenger - chemistry ; RNA, Messenger - genetics ; scFv ; Selection, Genetic ; Sequence Alignment ; Techniques ; Techniques used in virology ; Terminal protein ; Virology</subject><ispartof>Journal of immunological methods, 2004, Vol.284 (1), p.147-157</ispartof><rights>2003 Elsevier B.V.</rights><rights>2004 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c410t-cacfee2401dd244f4973764c05211f34eb4a8287e9e968adc58063db9162936b3</citedby><cites>FETCH-LOGICAL-c410t-cacfee2401dd244f4973764c05211f34eb4a8287e9e968adc58063db9162936b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jim.2003.10.009$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3536,4009,27902,27903,27904,45974</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=15414124$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/14736425$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lee, Myung-Shin</creatorcontrib><creatorcontrib>Kwon, Myung-Hee</creatorcontrib><creatorcontrib>Kim, Kyongmin Hwang</creatorcontrib><creatorcontrib>Shin, Ho-Joon</creatorcontrib><creatorcontrib>Park, Sun</creatorcontrib><creatorcontrib>Kim, Hyung-Il</creatorcontrib><title>Selection of scFvs specific for HBV DNA polymerase using ribosome display</title><title>Journal of immunological methods</title><addtitle>J Immunol Methods</addtitle><description>We applied a ribosome display technique to a mouse scFv library to select single chain variable fragments (scFvs) specific for the terminal protein (TP) of hepatitis B virus (HBV) DNA polymerase. Synthetic TP-peptide was used as an antigen to obtain scFvs that recognize specific epitopes within the TP domain, the priming site of HBV DNA polymerase. The scFv DNA library was transcribed in vitro to mRNA for ribosome display. scFv–ribosome–mRNA complexes were produced using a rabbit reticulocyte lysate system, and were panned against TP-peptide under appropriate conditions. After each panning, putative scFv-encoding genes were recovered by RT-PCR, and the analysis showed that scFv-ribosome-mRNA complexes were specifically selected by the TP-peptide. We used a radioimmunoassay to show that the TP-peptide-specific scFv pools were enriched through the selection process. Selected scFvs showed binding activity for both the TP-peptide and the HBV DNA polymerase protein in an ELISA. Sequence analysis showed that each TP-specific scFv had a different sequence, and that random mutagenesis was mediated by ribosome display.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Antibodies, Viral - blood</subject><subject>Antibody Specificity</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Chromatography, Affinity</subject><subject>DNA-Directed DNA Polymerase - genetics</subject><subject>DNA-Directed DNA Polymerase - immunology</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>Female</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fundamental immunology</subject><subject>HBV DNA polymerase</subject><subject>Hepatitis B virus</subject><subject>Hepatitis B virus - enzymology</subject><subject>Hepatitis B virus - genetics</subject><subject>Immunoglobulin Variable Region - genetics</subject><subject>Immunoglobulin Variable Region - immunology</subject><subject>Mice</subject><subject>Mice, Inbred BALB C</subject><subject>Microbiology</subject><subject>Molecular immunology</subject><subject>Molecular Sequence Data</subject><subject>Protein Biosynthesis - immunology</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Ribosome display</subject><subject>Ribosomes - immunology</subject><subject>RNA, Messenger - chemistry</subject><subject>RNA, Messenger - genetics</subject><subject>scFv</subject><subject>Selection, Genetic</subject><subject>Sequence Alignment</subject><subject>Techniques</subject><subject>Techniques used in virology</subject><subject>Terminal protein</subject><subject>Virology</subject><issn>0022-1759</issn><issn>1872-7905</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kE1r3DAQhkVIabZJf0AuRZf05u1Ilj9ETknaNIHQHvJxFbI8Klpsy9HsBvbfR8su5NbTMMPzvgwPY-cClgJE_WO1XIVxKQHKvC8B9BFbiLaRRaOhOmYLACkL0VT6hH0hWgGAgBo-sxOhmrJWslqw-0cc0K1DnHj0nNztG3Ga0QUfHPcx8bvrF_7zzxWf47AdMVlCvqEw_eMpdJHiiLwPNA92e8Y-eTsQfj3MU_Z8--vp5q54-Pv7_ubqoXBKwLpw1nlEqUD0vVTKK92UTa0cVFIIXyrslG1l26BGXbe2d1ULddl3WtRSl3VXnrLv-945xdcN0tqMgRwOg50wbsiIRpetanUGxR50KRIl9GZOYbRpawSYnT-zMtmf2fnbnbK_nPl2KN90I_YfiYOwDFwcAEvODj7ZyQX64CollJAqc5d7DrOKt4DJkAs4OexDyr5NH8N_3ngHRx2MAg</recordid><startdate>2004</startdate><enddate>2004</enddate><creator>Lee, Myung-Shin</creator><creator>Kwon, Myung-Hee</creator><creator>Kim, Kyongmin Hwang</creator><creator>Shin, Ho-Joon</creator><creator>Park, Sun</creator><creator>Kim, Hyung-Il</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>7T5</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope></search><sort><creationdate>2004</creationdate><title>Selection of scFvs specific for HBV DNA polymerase using ribosome display</title><author>Lee, Myung-Shin ; Kwon, Myung-Hee ; Kim, Kyongmin Hwang ; Shin, Ho-Joon ; Park, Sun ; Kim, Hyung-Il</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c410t-cacfee2401dd244f4973764c05211f34eb4a8287e9e968adc58063db9162936b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Antibodies, Viral - blood</topic><topic>Antibody Specificity</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Chromatography, Affinity</topic><topic>DNA-Directed DNA Polymerase - genetics</topic><topic>DNA-Directed DNA Polymerase - immunology</topic><topic>Enzyme-Linked Immunosorbent Assay</topic><topic>Female</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fundamental immunology</topic><topic>HBV DNA polymerase</topic><topic>Hepatitis B virus</topic><topic>Hepatitis B virus - enzymology</topic><topic>Hepatitis B virus - genetics</topic><topic>Immunoglobulin Variable Region - genetics</topic><topic>Immunoglobulin Variable Region - immunology</topic><topic>Mice</topic><topic>Mice, Inbred BALB C</topic><topic>Microbiology</topic><topic>Molecular immunology</topic><topic>Molecular Sequence Data</topic><topic>Protein Biosynthesis - immunology</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Ribosome display</topic><topic>Ribosomes - immunology</topic><topic>RNA, Messenger - chemistry</topic><topic>RNA, Messenger - genetics</topic><topic>scFv</topic><topic>Selection, Genetic</topic><topic>Sequence Alignment</topic><topic>Techniques</topic><topic>Techniques used in virology</topic><topic>Terminal protein</topic><topic>Virology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lee, Myung-Shin</creatorcontrib><creatorcontrib>Kwon, Myung-Hee</creatorcontrib><creatorcontrib>Kim, Kyongmin Hwang</creatorcontrib><creatorcontrib>Shin, Ho-Joon</creatorcontrib><creatorcontrib>Park, Sun</creatorcontrib><creatorcontrib>Kim, Hyung-Il</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Immunology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Journal of immunological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lee, Myung-Shin</au><au>Kwon, Myung-Hee</au><au>Kim, Kyongmin Hwang</au><au>Shin, Ho-Joon</au><au>Park, Sun</au><au>Kim, Hyung-Il</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Selection of scFvs specific for HBV DNA polymerase using ribosome display</atitle><jtitle>Journal of immunological methods</jtitle><addtitle>J Immunol Methods</addtitle><date>2004</date><risdate>2004</risdate><volume>284</volume><issue>1</issue><spage>147</spage><epage>157</epage><pages>147-157</pages><issn>0022-1759</issn><eissn>1872-7905</eissn><coden>JIMMBG</coden><abstract>We applied a ribosome display technique to a mouse scFv library to select single chain variable fragments (scFvs) specific for the terminal protein (TP) of hepatitis B virus (HBV) DNA polymerase. Synthetic TP-peptide was used as an antigen to obtain scFvs that recognize specific epitopes within the TP domain, the priming site of HBV DNA polymerase. The scFv DNA library was transcribed in vitro to mRNA for ribosome display. scFv–ribosome–mRNA complexes were produced using a rabbit reticulocyte lysate system, and were panned against TP-peptide under appropriate conditions. After each panning, putative scFv-encoding genes were recovered by RT-PCR, and the analysis showed that scFv-ribosome-mRNA complexes were specifically selected by the TP-peptide. We used a radioimmunoassay to show that the TP-peptide-specific scFv pools were enriched through the selection process. Selected scFvs showed binding activity for both the TP-peptide and the HBV DNA polymerase protein in an ELISA. Sequence analysis showed that each TP-specific scFv had a different sequence, and that random mutagenesis was mediated by ribosome display.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>14736425</pmid><doi>10.1016/j.jim.2003.10.009</doi><tpages>11</tpages></addata></record> |
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| subjects | Amino Acid Sequence Animals Antibodies, Viral - blood Antibody Specificity Base Sequence Biological and medical sciences Chromatography, Affinity DNA-Directed DNA Polymerase - genetics DNA-Directed DNA Polymerase - immunology Enzyme-Linked Immunosorbent Assay Female Fundamental and applied biological sciences. Psychology Fundamental immunology HBV DNA polymerase Hepatitis B virus Hepatitis B virus - enzymology Hepatitis B virus - genetics Immunoglobulin Variable Region - genetics Immunoglobulin Variable Region - immunology Mice Mice, Inbred BALB C Microbiology Molecular immunology Molecular Sequence Data Protein Biosynthesis - immunology Reverse Transcriptase Polymerase Chain Reaction Ribosome display Ribosomes - immunology RNA, Messenger - chemistry RNA, Messenger - genetics scFv Selection, Genetic Sequence Alignment Techniques Techniques used in virology Terminal protein Virology |
| title | Selection of scFvs specific for HBV DNA polymerase using ribosome display |
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