TY - JOUR
T1 - Prokaryotic expression, purification and immunogenicity in rabbits of the small antigen of hepatitis delta virus
AU - Tunitskaya, Vera L.
AU - Eliseeva, Olesja V.
AU - Valuev-Elliston, Vladimir T.
AU - Tyurina, Daria A.
AU - Zakirova, Natalia F.
AU - Khomich, Olga A.
AU - Kalis, Martins
AU - Latyshev, Oleg E.
AU - Starodubova, Elizaveta S.
AU - Ivanova, Olga N.
AU - Kochetkov, Sergey N.
AU - Isaguliants, Maria G.
AU - Ivanov, Alexander V.
N1 - Funding Information:
Expression and purification of HDV antigen was supported by Russian Foundation for Basic Research (grant 16-04-01490a). Evaluation of serum by Western blot and confocal microscopy was supported by Russian Science Foundation (grant 14-14-01021). Experiments in rabbits were supported by the Swedish Institute grants 09272_2013 and 19806_2016. Cross-border collaboration of the partners, exchange of the materials and standard operation procedures used in the study, and dissemination of the data were supported by the EU Twinning project VACTRAIN, contract nr 692293.
Publisher Copyright:
© 2016 by the authors; licensee MDPI, Basel, Switzerland.
PY - 2016/10/1
Y1 - 2016/10/1
N2 - 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.
AB - 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.
KW - Hepatitis delta virus
KW - Prokaryotic expression
KW - Protein purification
KW - Rabbit immunization
UR - http://www.scopus.com/inward/record.url?scp=84992117696&partnerID=8YFLogxK
U2 - 10.3390/ijms17101721
DO - 10.3390/ijms17101721
M3 - Article
C2 - 27775592
AN - SCOPUS:84992117696
SN - 1661-6596
VL - 17
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 10
M1 - 1721
ER -
