OBTAINING THE RECOMBINANT ORF3 PROTEIN OF HEPATITIS E GENOTYPE 3 AND EVALUATION OF ITS ANTIGENIC PROPERTIES

Cover Page


Cite item

Full Text

Abstract

Aim. Design аис1 construction of the hepatitis E virus (HEV) genotype 3 full-size ORF3 recombimnt polypeptide. Materials and methods. Escherichia coli strains, ptasmid vectors, serologiral and biological amples, molecular biological, bioinformatic, biotechnological, biochemical and serological methods.Results. RNA was isolated from pig fecal extracts collected on Belgorod farms and was used in RT-PCR to obtain the fragment of the orf3 gene of the hepatitis E virus genotype 3. Using A/T-cloning a recombinant plasmid was obtained with insertion of a DNA fragment (230 bp) encoding the N-terminal region of the ORF3 protein. The primary structure of the missing C-terminal region of the ORF3 VGE of the genotype 3 was calculated by bioinformatics methods. Codon optimization of the sequence for biosynthesis in E.coli cells was performed. For constructing the recombinant plasmid a chemically synthesized DNA fragment encoding the fulllength ORF3 protein had been used. E.coli strain producing full-size recombinant protein ORF3 fused to E.coli beta-galactosidase was developed. Recombinant protein ORF3 had been isolated from the inclusion bodies of the E.coli biomass and purified by size exclusion chromatography. Antigenic specificity of recombinant polypeptide had been confirmed in immunochemical reactions (ELISA, Western blot) with sera from patients with hepatitis E and control groups of patients. Conclusion. HEV genotype 3 ORF3 recombinant antigen had been designed, and itfs applicability in diagnostic tests had been experimentally confirmed.

About the authors

G. I. Alatortseva

Mechnikov Research Institute of Vaccines and Sera

Author for correspondence.
Russian Federation

A. V. Sidorov

Mechnikov Research Institute of Vaccines and Sera

Russian Federation

L. N. Nesterenko

Mechnikov Research Institute of Vaccines and Sera

Russian Federation

L. N. Luhverchik

Mechnikov Research Institute of Vaccines and Sera

Russian Federation

A. V. Milovanova

Mechnikov Research Institute of Vaccines and Sera

Russian Federation

Yu. I. Ammur

Mechnikov Research Institute of Vaccines and Sera

Russian Federation

M. I. Mikhailov

Mechnikov Research Institute of Vaccines and Sera, Russian Medical Academy of Postgraduate Education

Russian Federation

K. K. Kyuregyan

Mechnikov Research Institute of Vaccines and Sera, Russian Medical Academy of Postgraduate Education

Russian Federation

S. V. Zhavoronok

Belarusian State Medidl University

Russian Federation

V. V. Zverev

Mechnikov Research Institute of Vaccines and Sera

Russian Federation

References

  1. Алаторцев В.Е., Алаторцева Г.И. Патент РФ. 1992. № 2043409 на изобретение «Штамм бактерий Escherichia coli, используемый для получения рекомбинантных белков».
  2. Алаторцев В.Е., Алаторцева Г.И. Патент РФ.1992. № 2071501 на изобретение «Вектор pEL5a, предназначенный для экспрессии чужеродной ДНК».
  3. Алаторцева Г.И., Сидоров А.В., Нестеренко Л.Н. и.др. Получение рекомбинантного аналога гликопротеина Е вируса Varicella zoster: клонирование, экспрессия и исследование антигенных свойств. Эпидемиология и вакцинопрофилактика. 2016, 15, 1(86): 77-85.
  4. Алаторцева Г.И., Сидоров А.В., Нестеренко Л.Н. и др. Получение рекомбинантного белка ORF3 вируса гепатита Е 1 генотипа с применением метода оптимизации кодонов. Журн. микробиол. 2017, 6: 63-72.
  5. Алаторцева Г.И., Сидоров А.В., Нестеренко Л.Н. и др. Получение рекомбинантного аналога капсидного белка вируса гепатита Е 1: клонирование, экспрессия, очистка, оценка антигенных свойств. Журн. микробиол. 2017, 6: 72-80.
  6. Поляков А.Д. Особенности эпидемиологического процесса гепатита Е в Белгородской области. Научные ведомости. Сер. Медицина. Фармация, 2016, 5(226), вып. 33:79-83.
  7. Практическая химия белка. Ред. Дарбре А. М., 1989.
  8. Солонин С.А., Мальцева Н.С., Троценко О.Е. и др. Циркуляция вируса гепатита Е на территории Хабаровского края. Дальневосточный журнал инфекционной патологии. 2010, 16: 31-36.
  9. Altschul S., Gish W., Miller W. et al. Basic local alignment search too. J. Molecular. Biology. 1990, 215(3):403-410.
  10. Fogeda M., de Ory F., Aveltyn A. et al. Differential diagnosis ofhepatitis E virus, cytomegalovirus and Epstein-Barr virus infection in patients with suspected hepatitis E. J. Clin. Virol. 2009, 45(3): 259-261.
  11. Graff J., Torian U. et al. A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus. J. Virol. 2006, 80: 5919-5926.
  12. Hyams C., Mabayoje D.A., Copping R. et al. Serological cross reactivity to CMV and EBV causes problems in the diagnosis of acute hepatitis E virus infection. J. Med. Virol. 2014, 86(3): 478-483.
  13. Khudyakov Y.E., Khudyakova N.S., Fields H.A. et al. Epitope mapping in proteins of hepatitis E virus. Virology. 1993, 194(1): 89-96.
  14. Khudyakov Y. E., Khudyakova N.S, Jue D.L. et al. Comparative characterization of antigenic epitopes in the immunodominant region of the protein encoded by open reading frame 3 in Burmese and Mexican strains of hepatitis E virus. J. Gen. Virol. 1994, 75 (3): 641-646.
  15. Koonin E.V., Gorbalenya A.E., Purdy M.A. et al. Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses. Proc. Natl. Acad. Sci. USA. 1992, 89(17): 8259-8263.
  16. Ma H., Song X., Li Z. et al. Varying abilities of recombinant polypeptides from different regions of hepatitis E virus ORF2 and ORF3 to detect anti-HEV immunoglobulin M. J. Med. Virol. 2009, 81(6): 1052-1061.
  17. Obriadina A., Meng J.H., Ulanova T. et al. A new enzyme immunoassay for the detection of antibody to hepatitis E virus. J. Gastroenterol Hepatol. 2002, Suppl 3:360-364.
  18. Towbin H., Staehlin T., Gordon Y. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 1979, 76(9):4350-4359.
  19. Yarbough P.O., Tam A.W., Fry K.E. et al. Hepatitis E virus: identification of type-common epitopes. J. Virol. 1991, 659(11): 5790-5797.
  20. Zhou Y.H., Purcell R.H., Emerson S.U. An ELISA for putative neutralizing antibodies to hepatitis E virus detects antibodies to genotypes 1, 2, 3, and 4. Vaccine. 2004, 22(20): 2578-2585.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download

Copyright (c) 2018 Alatortseva G.I., Sidorov A.V., Nesterenko L.N., Luhverchik L.N., Milovanova A.V., Ammur Y.I., Mikhailov M.I., Kyuregyan K.K., Zhavoronok S.V., Zverev V.V.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
Регистрационный номер и дата принятия решения о регистрации СМИ: ПИ № ФС77-75442 от 01.04.2019 г.