Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus
Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commer...
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| creator | Tunitskaya, Vera L Eliseeva, Olesja V Valuev-Elliston, Vladimir T Tyurina, Daria A Zakirova, Natalia F Khomich, Olga A Kalis, Martins Latyshev, Oleg E Starodubova, Elizaveta S Ivanova, Olga N Kochetkov, Sergey N Isaguliants, Maria G Ivanov, Alexander V |
| description | Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in
of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 10⁷. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy. |
| doi_str_mv | 10.3390/ijms17101721 |
| format | Article |
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of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 10⁷. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.</description><identifier>ISSN: 1422-0067</identifier><identifier>ISSN: 1661-6596</identifier><identifier>EISSN: 1422-0067</identifier><identifier>DOI: 10.3390/ijms17101721</identifier><identifier>PMID: 27775592</identifier><language>eng</language><publisher>Switzerland: MDPI AG</publisher><subject>Animals ; Antibodies, Viral - biosynthesis ; Antibodies, Viral - blood ; Antibodies, Viral - immunology ; Cell Line ; Cloning, Molecular ; Communication ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Female ; Fluorescent Antibody Technique ; Hepatitis B virus ; Hepatitis D virus ; Hepatitis delta Antigens - biosynthesis ; Hepatitis delta Antigens - immunology ; Hepatitis delta virus ; Hepatitis Delta Virus - genetics ; Hepatitis Delta Virus - immunology ; Humans ; Immunization ; Protein expression ; Rabbits ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; RNA, Viral - genetics</subject><ispartof>International journal of molecular sciences, 2016-10, Vol.17 (10), p.1721-1721</ispartof><rights>Copyright MDPI AG 2016</rights><rights>2016 by the authors; licensee MDPI, Basel, Switzerland. 2016</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c445t-cdc6bb7606f2f297ade1b1d13ed8735e8d5761c6b38e52b9ccbdf89e6db5a3ac3</citedby><cites>FETCH-LOGICAL-c445t-cdc6bb7606f2f297ade1b1d13ed8735e8d5761c6b38e52b9ccbdf89e6db5a3ac3</cites><orcidid>0000-0002-5659-9679 ; 0000-0001-9382-2254</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085752/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085752/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27775592$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Tunitskaya, Vera L</creatorcontrib><creatorcontrib>Eliseeva, Olesja V</creatorcontrib><creatorcontrib>Valuev-Elliston, Vladimir T</creatorcontrib><creatorcontrib>Tyurina, Daria A</creatorcontrib><creatorcontrib>Zakirova, Natalia F</creatorcontrib><creatorcontrib>Khomich, Olga A</creatorcontrib><creatorcontrib>Kalis, Martins</creatorcontrib><creatorcontrib>Latyshev, Oleg E</creatorcontrib><creatorcontrib>Starodubova, Elizaveta S</creatorcontrib><creatorcontrib>Ivanova, Olga N</creatorcontrib><creatorcontrib>Kochetkov, Sergey N</creatorcontrib><creatorcontrib>Isaguliants, Maria G</creatorcontrib><creatorcontrib>Ivanov, Alexander V</creatorcontrib><title>Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus</title><title>International journal of molecular sciences</title><addtitle>Int J Mol Sci</addtitle><description>Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in
of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 10⁷. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.</description><subject>Animals</subject><subject>Antibodies, Viral - biosynthesis</subject><subject>Antibodies, Viral - blood</subject><subject>Antibodies, Viral - immunology</subject><subject>Cell Line</subject><subject>Cloning, Molecular</subject><subject>Communication</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Female</subject><subject>Fluorescent Antibody Technique</subject><subject>Hepatitis B virus</subject><subject>Hepatitis D virus</subject><subject>Hepatitis delta Antigens - biosynthesis</subject><subject>Hepatitis delta Antigens - immunology</subject><subject>Hepatitis delta virus</subject><subject>Hepatitis Delta Virus - genetics</subject><subject>Hepatitis Delta Virus - immunology</subject><subject>Humans</subject><subject>Immunization</subject><subject>Protein expression</subject><subject>Rabbits</subject><subject>Recombinant Proteins - 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Academic</collection><collection>Neurosciences Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>International journal of molecular sciences</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tunitskaya, Vera L</au><au>Eliseeva, Olesja V</au><au>Valuev-Elliston, Vladimir T</au><au>Tyurina, Daria A</au><au>Zakirova, Natalia F</au><au>Khomich, Olga A</au><au>Kalis, Martins</au><au>Latyshev, Oleg E</au><au>Starodubova, Elizaveta S</au><au>Ivanova, Olga N</au><au>Kochetkov, Sergey N</au><au>Isaguliants, Maria G</au><au>Ivanov, Alexander V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus</atitle><jtitle>International journal of molecular sciences</jtitle><addtitle>Int J Mol Sci</addtitle><date>2016-10-20</date><risdate>2016</risdate><volume>17</volume><issue>10</issue><spage>1721</spage><epage>1721</epage><pages>1721-1721</pages><issn>1422-0067</issn><issn>1661-6596</issn><eissn>1422-0067</eissn><abstract>Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in
of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 10⁷. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.</abstract><cop>Switzerland</cop><pub>MDPI AG</pub><pmid>27775592</pmid><doi>10.3390/ijms17101721</doi><tpages>1</tpages><orcidid>https://orcid.org/0000-0002-5659-9679</orcidid><orcidid>https://orcid.org/0000-0001-9382-2254</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Antibodies, Viral - biosynthesis Antibodies, Viral - blood Antibodies, Viral - immunology Cell Line Cloning, Molecular Communication Enzyme-Linked Immunosorbent Assay Escherichia coli Escherichia coli - genetics Escherichia coli - metabolism Female Fluorescent Antibody Technique Hepatitis B virus Hepatitis D virus Hepatitis delta Antigens - biosynthesis Hepatitis delta Antigens - immunology Hepatitis delta virus Hepatitis Delta Virus - genetics Hepatitis Delta Virus - immunology Humans Immunization Protein expression Rabbits Recombinant Proteins - genetics Recombinant Proteins - metabolism RNA, Viral - genetics |
| title | Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus |
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