title: Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus.
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 inE. coliof 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.
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. ve_tun@mail.ru.
authors: Vera L
authors: VL
authors: Tunitskaya
authors: Gamaleya Research Center of Epidemiology and Microbiology, Gamaleja str. 16, Moscow 123098, Russia. olesenka80@mail.ru.
authors: Olesja V
authors: OV
authors: Eliseeva
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. gansfaust@mail.ru.
authors: Vladimir T
authors: VT
authors: Valuev-Elliston
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. tyurina.dascha@gmail.com.
authors: Daria A
authors: DA
authors: Tyurina
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. nat_zakirova@mail.ru.
authors: Natalia F
authors: NF
authors: Zakirova
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. o.a.khomich@gmail.com.
authors: Olga A
authors: OA
authors: Khomich
authors: A Kirchenstein Institute of Microbiology and Virology, Research Department, Riga Stradins University, Dzirciema iela 16, Riga LV-1007, Latvia. martins.kalis@rsu.lv.
authors: Martins
authors: M
authors: Kalis
authors: Gamaleya Research Center of Epidemiology and Microbiology, Gamaleja str. 16, Moscow 123098, Russia. oleglat80@mail.ru.
authors: Oleg E
authors: OE
authors: Latyshev
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. estarodubova@yandex.ru.
authors: Elizaveta S
authors: ES
authors: Starodubova
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. olgaum@yandex.ru.
authors: Olga N
authors: ON
authors: Ivanova
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. kochet@eimb.ru.
authors: Sergey N
authors: SN
authors: Kochetkov
authors: Gamaleya Research Center of Epidemiology and Microbiology, Gamaleja str. 16, Moscow 123098, Russia. maria.issagouliantis@rsu.lv.|A Kirchenstein Institute of Microbiology and Virology, Research Department, Riga Stradins University, Dzirciema iela 16, Riga LV-1007, Latvia. maria.issagouliantis@rsu.lv.
authors: Maria G
authors: MG
authors: Isaguliants
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia. aivanov@yandex.ru.
authors: Alexander V
authors: AV
authors: Ivanov
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000097:blood / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000276:immunology*; D035923:Hepatitis delta Antigens / Q000096:biosynthesis / Q000276:immunology*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D035923:Hepatitis delta Antigens; D012367:RNA, Viral; D011994:Recombinant Proteins
keywords: hepatitis delta virus; prokaryotic expression; protein purification; rabbit immunization