veterinaryВетеринария сегодняVeterinary Science Today2304-196X2658-6959"Veinard"10.29326/2304-196X-2018-2-25-3-7veterinary-353Research ArticleБОЛЕЗНИ СВИНЕЙPORCINE DISEASESКЛОНИРОВАНИЕ ГЕНОВ, КОДИРУЮЩИХ ТРАНСМЕМБРАННЫЕ БЕЛКИ И БЕЛКИ, ОТВЕТСТВЕННЫЕ ЗА ВИРУЛЕНТНОСТЬ ВИРУСА АФРИКАНСКОЙ ЧУМЫ СВИНЕЙCLONING OF GENES ENCODING TRANSMEMBRANE PROTEINS AND PROTEINS RESPONSIBLE FOR AFRICAN SWINE FEVER VIRUS VIRULENCEМазлумАлиMazloumAli

Аспирант.

Владимир

PhD Student, 

Vladimir

ali.mazloum6@gmail.comЗиняковН. Г.ZinyakovN. G.

Научный сотрудник, кандидат биологических наук.

 Владимир

 Researcher, Candidate of Science (Biology), 

Vladimir

zinyakov@arriah.ruИголкинА. С.IgolkinA. S.

Заведующий лабораторией, кандидат ветеринарных наук.

Head ofLaboratory, Candidate of Science (Veterinary Medicine), 

Vladimir

igolkin_as@arriah.ruВласоваН. Н.VlasovaN. N.

Главный научный сотрудник, доктор биологических наук, 

г. Владимир 

Senior Researcher, Doctor of Science (Biology), 

Vladimir

vlasova_nn@arriah.ruФГБУ «ВНИИЗЖ», г. ВладимирРоссияFGBI "ARRIAH", VladimirRussian Federation2018020720180237Copyright © Мазлум А., Зиняков Н.Г., Иголкин А.С., Власова Н.Н., 20182018Мазлум А., Зиняков Н.Г., Иголкин А.С., Власова Н.Н.Mazloum A., Zinyakov N.G., Igolkin A.S., Vlasova N.N.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://veterinary.arriah.ru/jour/article/view/353

Представлены результаты клонирования генов X69R, A179L, E248R, I215L и DP96R изолята Krasnodar 07/17 вируса африканской чумы свиней и анализ их нуклеотидных последовательностей. Полученные клоны пополнили ранее созданную клонотеку, содержащую клоны с 8 генами изолята Krasnodar 06/12. Клоны, содержащие гены X69R, A179L, E248R, I215L и DP96R изолята Krasnodar 07/17 вируса африканской чумы свиней, будут использованы для получения рекомбинантных белков и изучения их влияния на репродукцию вируса in vitro, их роли в инфекционности вируса, степени выраженности клинических признаков и вирулентности. В работе использовали прокариотический вектор pJET1.2/blunt. Таким образом, имеющаяся в референтной лаборатории по африканской чуме свиней ФГБУ «ВНИИЗЖ» клонотека дополнена плазмидными конструкциями pJET1.2-X69R, pJET1.2-A179L, pJET1.2-E248R, pJET1.2I215L и pJET1.2-DP96R, несущими 5 генов изолята Krasnodar 07/17 вируса африканской чумы свиней. Доля клонированных генов вируса составила 3,01% генома изолята Krasnodar 07/17, следовательно, общий объем клонотеки достиг 7,82%.

Results of cloning X69R, A179L, E248R, I215L and DP96R genes of ASF virus Krasnodar 07/17 isolate and analysis of their nucleotide sequences are presented. Obtained clones were added to the previously constructed clone library comprising clones of 8 genes of Krasnodar 06/12 isolate. Clones containing X69R, A179L, E248R, I215L and DP96R genes of ASF virus Krasnodar 07/17 isolate will be used for recombinant protein obtaining and testing for their effect on in vitro virus reproduction and their role in the virus infectivity, level of clinical manifestations and virulence. Prokaryotic vector, pJET1.2/ blunt, was used. Thus, the clone library available at the FGBI “ARRIAH” Reference Laboratory for African swine fever was supplemented by pJET1.2-X69R, pJET1.2-A179L, pJET1.2-E248R, pJET1.2-I215L and pJET1.2-DP96R plasmid constructions containing 5 genes of ASF virus Krasnodar 07/17 isolate. Proportion of cloned virus genes was 3.01% of Krasnodar 07/17 isolate genome, hence, total amount of the clone library has reached 7.82%.

африканская чума свиней (АЧС)трансмембранные белкибелкиответственные за вирулентность вируса АЧСсеквенированиеклонотека геновAfrican swine fever (ASF)transmembrane proteinsproteins responsible for ASF virus virulencesequencinggene clone libraryReferencesИнструкция по применению комплекта реагентов для экстракции ДНК из клинического материала «АмплиПрайм ДНК-сорб-В». – URL: http://www.interlabservice.ru/upload/iblock/ff0/Инструкция%20ДНК-сорб-В_30.10.2012.pdf.Instruction for use of AmpliPrime-DNA-Sorb-B nucleic acid extraction kit [Instrukcija po primeneniju komplekta reagentov dlja jekstrakcii DNK iz klinicheskogo materiala «AmpliPrajm DNK-sorbV»]. – URL: URL: http://www.interlabservice.ru/upload/iblock/ff0/ Инструкция%20ДНК-сорб-В_30.10.2012.pdf (in Russian).Конструирование библиотеки генов, кодирующих иммунологически значимые белки вируса АЧС изолята Кrasnodar 06/12 / А. А. Варенцова, И. В. Шевченко, Н. Г. Зиняков [и др.] // Актуальные проблемы гуманитарных и естественных наук. – 2014. – № 5–1. – С. 28–35.Varentsova А. А., Shevchenko I. V., Zinyakov N. G. et al. Construction of library of genes encoding immunologically significant proteins of ASF virus Кrasnodar 06/12 isolate [Konstruirovanie biblioteki genov, kodirujushhih immunologicheski znachimye belki virusa AChS izoljata Krasnodar 06/12]. Current aspects of humanities and the sciences. 2014; 5–1: 28–35 (in Russian).A nonessential African swine fever virus gene UK is a significant virulence determinant in domestic swine / L. Zsak, E. Caler, Z. Lu [et al.] // J. Virol. – 1998. – Vol. 72, No. 2. – P. 1028–1035.Zsak L., Caler E., Lu Z. et al. A nonessential African swine fever virus gene UK is a significant virulence determinant in domestic swine. J. Virol. 1998; 72 (2): 1028–1035. 4. Brun A., Rivas C., Esteban M. et al. African swine fever virus gene A179L, a viral homologue of bcl-2, protects cells from programmed cell death. Virology. 1996; 225 (1): 227–230.African swine fever virus gene A179L, a viral homologue of bcl-2, protects cells from programmed cell death / A. Brun, C. Rivas, M. Esteban [et al.] // Virology. – 1996. – Vol. 225, No. 1. – P. 227–230.Rodríguez J. M., García-Escudero R., Salas M. L., Andrés G. African swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites. J. Virol. 2004; 78 (8): 4299–4313. DOI: 10.1128/JVI.78.8.4299-4313.2004.African swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites / J. M. Rodríguez, R. García-Escudero, M. L. Salas, G. Andrés // J. Virol. – 2004. – Vol. 78, No. 8. – P. 4299–4313. DOI: 10.1128/JVI.78.8.4299–4313.2004.Neilan J. G., Lu Z., Afonso C. L. et al. An African swine fever virus gene with similarity to the proto-oncogene bcl-2 and the Epstein-Barr virus gene BHRF1. J. Virol. 1993: 67 (7): 4391–4394.An African swine fever virus gene with similarity to the proto-oncogene bcl-2 and the Epstein-Barr virus gene BHRF1 / J. G. Neilan, Z. Lu, C. L. Afonso [et al.] // J. Virol. – 1993. – Vol. 67, No. 7. – P. 4391–4394.Stewart M. D., Ritterhoff T., Klevit R. E., Brzovic P. S. E2 enzymes: More than just middlemen. Cell Res. 2016: 26 (4): 423–440. DOI: 10.1038/cr.2016.35.E2 enzymes: More than just middlemen / M. D. Stewart, T. Ritterhoff, R. E. Klevit, P. S. Brzovic // Cell Res. – 2016. – Vol. 26, No. 4. – P. 423–440. DOI: 10.1038/cr.2016.35.He Y., Xiang Z., Mobley H. L. T. Vaxign: The first web-based vaccine design program for reverse vaccinology and applications for vaccine development. J. Biomed. Biotechnol. Vol. 2010. – URL: http://dx.doi.org/10.1155/2010/297505.He Y., Xiang Z., Mobley H. L. T. Vaxign: The first web-based vaccine design program for reverse vaccinology and applications for vaccine development // J. Biomed. Biotechnol. – Vol. 2010. – URL: http://dx.doi.org/10.1155/2010/297505.Kollnberger S. D., Gutierrez-Castañeda B., Foster-Cuevas M. et al. Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody. J. Gen. Virol. 2002; 83: 1331–1342.Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody / S. D. Kollnberger, B. Gutierrez-Castañeda, M. Foster-Cuevas [et al.] // J. Gen. Virol. – 2002. – Vol. 83. – P. 1331–1342.Revilla Y., Cebrián A., Baixerás E. et al. Inhibition of apoptosis by the African swine fever virus bcl-2 homologue: role of the BH1 domain. Virology. 1997; 228 (2): 400–404.Inhibition of apoptosis by the African swine fever virus bcl-2 homologue: role of the BH1 domain / Y. Revilla, A. Cebrián, E. Baixerás [et al.] // Virology. – 1997. – Vol. 228, No. 2. – P. 400–404.Menon R., Farina C. Shared molecular and functional frameworks among five complex human disorders: a comparative study on interactomes linked to susceptibility genes. PLoS ONE. 2011; 6 (4):e18660. – URL: https://doi.org/10.1371/journal.pone.0018660.Menon R., Farina C. Shared molecular and functional frameworks among five complex human disorders: a comparative study on interactomes linked to susceptibility genes // PLoS One. – 2011. – Vol. 6 (4):e18660. – URL: https://doi.org/10.1371/journal.pone.0018660.Sanger F., Air G. M., Barrell B. G. et al. Nucleotide sequence of bacteriophage phi X174 DNA. Nature. 1977; 265 (5596): 687–695.Nucleotide sequence of bacteriophage phi X174 DNA / F. Sanger, G. M. Air, B. G. Barrell [et al.] // Nature. – 1977. – Vol. 265, No. 5596. – P. 687–695.Product Information Thermo Scientific CloneJET PCR Cloning Kit. – URL: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012966_CloneJET_PCR_Cloning_40rxn_UG.pdf.Product Information Thermo Scientific CloneJET PCR Cloning Kit. – URL: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012966_CloneJET_PCR_Cloning_40rxn_UG.pdf.QIAquick Gel Extraction Kit Protocol. – URL: http://www.indiana.edu/~lchenlab/protocol_files/agarose_gel_extraction.pdf.QIAquick Gel Extraction Kit Protocol. – URL: http://www.indiana.edu/~lchenlab/protocol_files/agarose_gel_extraction.pdf.Rodriguez J. M., Salas M. L., Viñuela E. Genes homologous to ubiquitin-conjugating proteins and eukaryotic transcription factor SII in African swine fever virus. Virology. 1992; 186 (1): 40–52.Rodriguez J. M., Salas M. L., Viñuela E. Genes homologous to ubiquitin-conjugating proteins and eukaryotic transcription factor SII in African swine fever virus // Virology. – 1992. – Vol. 186, No. 1. – P. 40–52.Lopera-Madrid J., Osorio J. E., He Y. et al. Safety and immunogenicity of mammalian cell-derived and Modified Vaccinia Ankara vectored African swine fever subunit antigens in swine. Vet. Immunol. Immunopathol. 2017; 185: 20–33. DOI: 10.1016/j.vetimm.2017.01.004.Safety and immunogenicity of mammalian cell-derived and Modified Vaccinia Ankara vectored African swine fever subunit antigens in swine / J. Lopera-Madrid, J. E. Osorio, Y. He [et al.] // Vet. Immunol. Immunopathol. – 2017. – Vol. 185. – P. 20–33. DOI: 10.1016/j.vetimm.2017.01.004.Xiang Z., He Y. Genome-wide prediction of vaccine targets for human herpes simplex viruses using Vaxign reverse vaccinology. BMC Bioinformatics. 2013; 14 (Suppl 4):S2. – URL: http://www.biomedcentral.com/1471-2105/14/S4/S2.Xiang Z., He Y. Genome-wide prediction of vaccine targets for human herpes simplex viruses using Vaxign reverse vaccinology // BMC Bioinformatics. – 2013. – Vol. 14 (Suppl 4):S2. – URL: http://www.biomedcentral.com/1471-2105/14/S4/S2.Xiang Z., He Y. Genome-wide prediction of vaccine targets for human herpes simplex viruses using Vaxign reverse vaccinology // BMC Bioinformatics. – 2013. – Vol. 14 (Suppl 4):S2. – URL: http://www.biomedcentral.com/1471-2105/14/S4/S2.

The authors declare that there are no conflicts of interest present.