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Potential Susceptibility Mutations in C Gene for Hepatitis B-Related Hepatocellular Carcinoma Identified by a Two-Stage Study in Qidong, China
Open AccessCommunication

Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia
Gamaleya Research Center of Epidemiology and Microbiology, Gamaleja str. 16, Moscow 123098, Russia
A Kirchenstein Institute of Microbiology and Virology, Research Department, Riga Stradins University, Dzirciema iela 16, Riga LV-1007, Latvia
Authors to whom correspondence should be addressed.
Academic Editor: Tatsuo Kanda
Int. J. Mol. Sci. 2016, 17(10), 1721;
Received: 25 August 2016 / Revised: 20 September 2016 / Accepted: 27 September 2016 / Published: 20 October 2016
(This article belongs to the Special Issue Hepatitis Virus Infection and Research)
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 E. coli 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. View Full-Text
Keywords: hepatitis delta virus; prokaryotic expression; protein purification; rabbit immunization hepatitis delta virus; prokaryotic expression; protein purification; rabbit immunization
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Tunitskaya, V.L.; Eliseeva, O.V.; Valuev-Elliston, V.T.; Tyurina, D.A.; Zakirova, N.F.; Khomich, O.A.; Kalis, M.; Latyshev, O.E.; Starodubova, E.S.; Ivanova, O.N.; Kochetkov, S.N.; Isaguliants, M.G.; Ivanov, A.V. Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus. Int. J. Mol. Sci. 2016, 17, 1721.

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