Expression and Characterization of Bluetongue Virus Serotype 21 VP7 Antigen: C-Terminal Truncated Protein has Significantly Reduced Antigenicity
In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide seque...
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| Veröffentlicht in: | Journal of Veterinary Medical Science 2011, Vol.73(5), pp.609-613 |
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| creator | HOSAMANI, Madhusudan SHIMIZU, Shinya HIROTA, Jiro KOKUHO, Takehiro KUBOTA, Takayuki WATANABE, Satoko OHTA, Masato MUNETA, Yoshihiro INUMARU, Shigeki |
| description | In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation. |
| doi_str_mv | 10.1292/jvms.10-0213 |
| format | Article |
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Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. 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Vet. Med. Sci.</addtitle><description>In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.</description><subject>Animals</subject><subject>Antigens, Viral - genetics</subject><subject>Antigens, Viral - metabolism</subject><subject>Baculovirus</subject><subject>Bluetongue - epidemiology</subject><subject>Bluetongue - virology</subject><subject>Bluetongue virus</subject><subject>Bluetongue virus - classification</subject><subject>Bluetongue virus - genetics</subject><subject>Bluetongue virus - immunology</subject><subject>Bluetongue virus - metabolism</subject><subject>Cattle</subject><subject>Cattle Diseases - epidemiology</subject><subject>Cattle Diseases - virology</subject><subject>Cell Line</subject><subject>Cricetinae</subject><subject>Gene Expression Regulation, Viral - physiology</subject><subject>Japan - epidemiology</subject><subject>serotype 21</subject><subject>Serotyping</subject><subject>Viral Core Proteins - genetics</subject><subject>Viral Core Proteins - metabolism</subject><subject>VP7</subject><issn>0916-7250</issn><issn>1347-7439</issn><issn>1347-7439</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0UFv2yAYBmA0rVqzbredJ2671B1gDGa3NurWSpVadVmvFsYfKZGNM8DTsl_RnzysZLnuAvrg-d7Li9AHSi4oU-zz5tcQLygpCKPlK7SgJZeF5KV6jRZEUVFIVpFT9DbGDcmEC_UGnTJKaylqvkAv17-3AWJ0o8fad3j5rIM2CYL7o9P8OFp81U-QRr-eAD-5MEX8HcKYdlvAjOKnB4kvfXJr8F_wslhBGJzXPV6FyRudoMMPGYPz-FnnTbf2zjqjfep3-BG6yWRx2HfGpd07dGJ1H-H94T5DP75er5Y3xd39t9vl5V1hKs5T0RLKOdFAKstqwStLlOFWKC6N7ZRpGWlBkVbaWgnOW5sn2bay48Ja0wlTnqFP-9xtGH9OEFMzuGig77WHcYqNIqzkNRXlf2UtlFRUlnWW53tpwhhjANtsgxt02DWUNHNZzVzWPMxlZf7xEDy1A3RH_K-dDK72YBOTXsMR6JCc6WGfJsummo9D6vHT5CYb8OVfDFWqug</recordid><startdate>2011</startdate><enddate>2011</enddate><creator>HOSAMANI, Madhusudan</creator><creator>SHIMIZU, Shinya</creator><creator>HIROTA, Jiro</creator><creator>KOKUHO, Takehiro</creator><creator>KUBOTA, Takayuki</creator><creator>WATANABE, Satoko</creator><creator>OHTA, Masato</creator><creator>MUNETA, Yoshihiro</creator><creator>INUMARU, Shigeki</creator><general>JAPANESE SOCIETY OF VETERINARY SCIENCE</general><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>7X8</scope><scope>7U9</scope><scope>H94</scope></search><sort><creationdate>2011</creationdate><title>Expression and Characterization of Bluetongue Virus Serotype 21 VP7 Antigen: C-Terminal Truncated Protein has Significantly Reduced Antigenicity</title><author>HOSAMANI, Madhusudan ; SHIMIZU, Shinya ; HIROTA, Jiro ; KOKUHO, Takehiro ; KUBOTA, Takayuki ; WATANABE, Satoko ; OHTA, Masato ; MUNETA, Yoshihiro ; INUMARU, Shigeki</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c544t-b01440ae05f28645f09c4f6947cfd9cb20be90b7f89644bfbe97bb7d46ffcd6c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Animals</topic><topic>Antigens, Viral - genetics</topic><topic>Antigens, Viral - metabolism</topic><topic>Baculovirus</topic><topic>Bluetongue - epidemiology</topic><topic>Bluetongue - virology</topic><topic>Bluetongue virus</topic><topic>Bluetongue virus - classification</topic><topic>Bluetongue virus - genetics</topic><topic>Bluetongue virus - immunology</topic><topic>Bluetongue virus - metabolism</topic><topic>Cattle</topic><topic>Cattle Diseases - epidemiology</topic><topic>Cattle Diseases - virology</topic><topic>Cell Line</topic><topic>Cricetinae</topic><topic>Gene Expression Regulation, Viral - physiology</topic><topic>Japan - epidemiology</topic><topic>serotype 21</topic><topic>Serotyping</topic><topic>Viral Core Proteins - genetics</topic><topic>Viral Core Proteins - metabolism</topic><topic>VP7</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>HOSAMANI, Madhusudan</creatorcontrib><creatorcontrib>SHIMIZU, Shinya</creatorcontrib><creatorcontrib>HIROTA, Jiro</creatorcontrib><creatorcontrib>KOKUHO, Takehiro</creatorcontrib><creatorcontrib>KUBOTA, Takayuki</creatorcontrib><creatorcontrib>WATANABE, Satoko</creatorcontrib><creatorcontrib>OHTA, Masato</creatorcontrib><creatorcontrib>MUNETA, Yoshihiro</creatorcontrib><creatorcontrib>INUMARU, Shigeki</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><jtitle>Journal of Veterinary Medical Science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>HOSAMANI, Madhusudan</au><au>SHIMIZU, Shinya</au><au>HIROTA, Jiro</au><au>KOKUHO, Takehiro</au><au>KUBOTA, Takayuki</au><au>WATANABE, Satoko</au><au>OHTA, Masato</au><au>MUNETA, Yoshihiro</au><au>INUMARU, Shigeki</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression and Characterization of Bluetongue Virus Serotype 21 VP7 Antigen: C-Terminal Truncated Protein has Significantly Reduced Antigenicity</atitle><jtitle>Journal of Veterinary Medical Science</jtitle><addtitle>J. Vet. Med. Sci.</addtitle><date>2011</date><risdate>2011</risdate><volume>73</volume><issue>5</issue><spage>609</spage><epage>613</epage><pages>609-613</pages><issn>0916-7250</issn><issn>1347-7439</issn><eissn>1347-7439</eissn><abstract>In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.</abstract><cop>Japan</cop><pub>JAPANESE SOCIETY OF VETERINARY SCIENCE</pub><pmid>21187684</pmid><doi>10.1292/jvms.10-0213</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Journal of Veterinary Medical Science, 2011, Vol.73(5), pp.609-613 |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; J-STAGE (Japan Science & Technology Information Aggregator, Electronic) Freely Available Titles - Japanese |
| subjects | Animals Antigens, Viral - genetics Antigens, Viral - metabolism Baculovirus Bluetongue - epidemiology Bluetongue - virology Bluetongue virus Bluetongue virus - classification Bluetongue virus - genetics Bluetongue virus - immunology Bluetongue virus - metabolism Cattle Cattle Diseases - epidemiology Cattle Diseases - virology Cell Line Cricetinae Gene Expression Regulation, Viral - physiology Japan - epidemiology serotype 21 Serotyping Viral Core Proteins - genetics Viral Core Proteins - metabolism VP7 |
| title | Expression and Characterization of Bluetongue Virus Serotype 21 VP7 Antigen: C-Terminal Truncated Protein has Significantly Reduced Antigenicity |
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