OBTAINING THE RECOMBINANT ORF3 PROTEIN OF HEPATITIS E GENOTYPE 3 AND EVALUATION OF ITS ANTIGENIC PROPERTIES
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 metho...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | Russian |
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Central Research Institute for Epidemiology
2018-10-01
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| Series: | Журнал микробиологии, эпидемиологии и иммунобиологии |
| Subjects: | |
| Online Access: | https://microbiol.elpub.ru/jour/article/view/307 |
| _version_ | 1798036248073338880 |
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| author | G. I. Alatortseva A. V. Sidorov L. N. Nesterenko L. N. Luhverchik A. V. Milovanova Yu. I. Ammur M. I. Mikhailov K. K. Kyuregyan S. V. Zhavoronok V. V. Zverev |
| author_facet | G. I. Alatortseva A. V. Sidorov L. N. Nesterenko L. N. Luhverchik A. V. Milovanova Yu. I. Ammur M. I. Mikhailov K. K. Kyuregyan S. V. Zhavoronok V. V. Zverev |
| author_sort | G. I. Alatortseva |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T21:10:03Z |
| format | Article |
| id | doaj.art-97c0c94eb9ea4209abe73a62487f90c4 |
| institution | Directory Open Access Journal |
| issn | 0372-9311 2686-7613 |
| language | Russian |
| last_indexed | 2024-04-11T21:10:03Z |
| publishDate | 2018-10-01 |
| publisher | Central Research Institute for Epidemiology |
| record_format | Article |
| series | Журнал микробиологии, эпидемиологии и иммунобиологии |
| spelling | doaj.art-97c0c94eb9ea4209abe73a62487f90c42022-12-22T04:03:04ZrusCentral Research Institute for EpidemiologyЖурнал микробиологии, эпидемиологии и иммунобиологии0372-93112686-76132018-10-0105465310.36233/0372-9311-2018-5-46-53307OBTAINING THE RECOMBINANT ORF3 PROTEIN OF HEPATITIS E GENOTYPE 3 AND EVALUATION OF ITS ANTIGENIC PROPERTIESG. I. Alatortseva0A. V. Sidorov1L. N. Nesterenko2L. N. Luhverchik3A. V. Milovanova4Yu. I. Ammur5M. I. Mikhailov6K. K. Kyuregyan7S. V. Zhavoronok8V. V. Zverev9Mechnikov Research Institute of Vaccines and SeraMechnikov Research Institute of Vaccines and SeraMechnikov Research Institute of Vaccines and SeraMechnikov Research Institute of Vaccines and SeraMechnikov Research Institute of Vaccines and SeraMechnikov Research Institute of Vaccines and SeraMechnikov Research Institute of Vaccines and Sera, Russian Medical Academy of Postgraduate EducationMechnikov Research Institute of Vaccines and Sera, Russian Medical Academy of Postgraduate EducationBelarusian State Medidl UniversityMechnikov Research Institute of Vaccines and SeraAim. 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.https://microbiol.elpub.ru/jour/article/view/307hepatitis e virusgenotype 3 hevorf3 generecombinant orf3 antigen, elisa, western blot |
| spellingShingle | G. I. Alatortseva A. V. Sidorov L. N. Nesterenko L. N. Luhverchik A. V. Milovanova Yu. I. Ammur M. I. Mikhailov K. K. Kyuregyan S. V. Zhavoronok V. V. Zverev OBTAINING THE RECOMBINANT ORF3 PROTEIN OF HEPATITIS E GENOTYPE 3 AND EVALUATION OF ITS ANTIGENIC PROPERTIES Журнал микробиологии, эпидемиологии и иммунобиологии hepatitis e virus genotype 3 hev orf3 gene recombinant orf3 antigen, elisa, western blot |
| title | OBTAINING THE RECOMBINANT ORF3 PROTEIN OF HEPATITIS E GENOTYPE 3 AND EVALUATION OF ITS ANTIGENIC PROPERTIES |
| title_full | OBTAINING THE RECOMBINANT ORF3 PROTEIN OF HEPATITIS E GENOTYPE 3 AND EVALUATION OF ITS ANTIGENIC PROPERTIES |
| title_fullStr | OBTAINING THE RECOMBINANT ORF3 PROTEIN OF HEPATITIS E GENOTYPE 3 AND EVALUATION OF ITS ANTIGENIC PROPERTIES |
| title_full_unstemmed | OBTAINING THE RECOMBINANT ORF3 PROTEIN OF HEPATITIS E GENOTYPE 3 AND EVALUATION OF ITS ANTIGENIC PROPERTIES |
| title_short | OBTAINING THE RECOMBINANT ORF3 PROTEIN OF HEPATITIS E GENOTYPE 3 AND EVALUATION OF ITS ANTIGENIC PROPERTIES |
| title_sort | obtaining the recombinant orf3 protein of hepatitis e genotype 3 and evaluation of its antigenic properties |
| topic | hepatitis e virus genotype 3 hev orf3 gene recombinant orf3 antigen, elisa, western blot |
| url | https://microbiol.elpub.ru/jour/article/view/307 |
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