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Journal for Veterinary Medicine, Biotechnology and Biosafety

Volume 7, Issue 3, September 2021, Pages 24–31

ISSN 2411-3174 (print version) ISSN 2411-0388 (online version)

DEVELOPMENT OF RECOMBINANT ANTIGEN EXPRESSION AND PURIFICATION FOR AFRICAN SWINE FEVER SEROLOGICAL DIAGNOSTICS

Kit M. Yu.

National Scientific Center ‘Institute of Experimental and Clinical Veterinary Medicine’, Kharkiv, Ukraine, e-mail: maryna_kit@ukr.net

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Citation for print version: Kit, M. Yu. (2021) ‘Development of recombinant antigen expression and purification for African swine fever serological diagnostics’, Journal for Veterinary Medicine, Biotechnology and Biosafety, 7(3), pp. 24–31.

Download PDF (online version)

Citation for online version: Kit, M. Yu. (2021) ‘Development of recombinant antigen expression and purification for African swine fever serological diagnostics’, Journal for Veterinary Medicine, Biotechnology and Biosafety. [Online] 7(3), pp. 24–31. DOI: 10.36016/JVMBBS-2021-7-3-4.

Summary. The paper reports the purification and its optimization of recombinant proteins p10, p32, p54, p54ΔTM, DNA ligase and DNA ligaseΔDBD of African swine fever virus. The corresponding coding sequences were subcloned into pASG-IBA105 and pASG-IBA103 vectors, multiplied and used for transformation of competent E. coli expression strain. Expressed proteins were purified using Strep-Tactin XT purification system under native and denaturing conditions, as well as using detergents according to the optimized protocol for recombinant proteins solubilization from inclusion bodies. Among all expressed and purified proteins p32 and p54 were found to be immunoreactive and specific. Although p54 was unstable during long-term storage, after further storage condition optimization, the protein can be used for indirect ASF ELISA development. Recombinant p32 was shown to be an effective antigen for ASF ELISA providing detection of antibodies against ASFV with low background signal

Keywords: ELISA, p10, p32, p54, DNA ligase

References:

Al Dahouk, S., Nöckler, K., Tomaso, H., Splettstoesser, W. D., Jungersen, G., Riber, U., Petry, T., Hoffmann, D., Scholz, H. C., Hensel, A. and Neubauer, H. (2005) ‘Seroprevalence of Brucellosis, Tularemia, and Yersiniosis in wild boars (Sus scrofa) from North-Eastern Germany’, Journal of Veterinary Medicine. Series B, 52(10), pp. 444–455. doi: https://doi.org/10.1111/j.1439-0450.2005.00898.x.

Alonso, C., Borca, M., Dixon, L., Revilla, Y., Rodriguez, F., Escribano, J. M. and ICTV Report Consortium (2018) ‘ICTV virus taxonomy profile: Asfarviridae’, Journal of General Virology, 99(5), pp. 613–614. doi: https://doi.org/10.1099/jgv.0.001049.

Basile, G. and Peticca, M. (2009) ‘Recombinant protein expression in Leishmania tarentolae’, Molecular Biotechnology, 43(3), pp. 273–278. doi: https://doi.org/10.1007/s12033-009-9213-5.

Beltrán-Alcrudo, D., Arias, M., Gallardo, C., Kramer, S. and Penrith, M. L. (2017) African Swine Fever: Detection and Diagnosis — A Manual for Veterinarians. FAO Animal Production and Health Manual, 19. Rome: FAO. Available at: http://www.fao.org/3/i7228en/I7228EN.pdf.

Burgess, R. R. (1996) ‘[12] Purification of overproduced Escherichia coli RNA polymerase σ factors by solubilizing inclusion bodies and refolding from Sarkosyl’, in Adhya, S. (ed.) RNA Polymerase and Associated Factors. Part A (Methods in Enzymology, 273). San Diego: Academic Press, pp. 145–149. doi: https://doi.org/10.1016/S0076-6879(96)73014-8.

Coligan, J. E. (1998) ‘Appendix 1B. Commonly Used Detergents’, Current Protocols in Protein Science, 11(1), pp. A.1B.1-A.1B.3. doi: https://doi.org/10.1002/0471140864.psa01bs11.

Dixon, L. K., Alonso, C., Escribano, J. M., Martins, C., Revilla, Y., Salas, M. L. and Takamatsu, H. (2012) ‘Asfarviridae’, in King, A. M. Q., Adams, M. J., Carstens, E. B. and Lefkowitz, E. J. (eds.) Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press, pp. 153–162. doi: https://doi.org/10.1016/B978-0-12-384684-6.00012-4.

Dixon, L. K., Stahl, K., Jori, F., Vial, L. and Pfeiffer, D. U. (2020) ‘African swine fever epidemiology and control’, Annual Review of Animal Biosciences, 8(1), pp. 221–246. doi: https://doi.org/10.1146/annurev-animal-021419-083741.

Heimerman, M. E., Murgia, M. V., Wu, P., Lowe, A. D., Jia, W. and Rowland, R. R. (2018) ‘Linear epitopes in African swine fever virus p72 recognized by monoclonal antibodies prepared against baculovirus-expressed antigen’, Journal of Veterinary Diagnostic Investigation, 30(3), pp. 406–412. doi: https://doi.org/10.1177/1040638717753966.

Ikonomou, L., Schneider, Y.-J. and Agathos, S. N. (2003) ‘Insect cell culture for industrial production of recombinant proteins’, Applied Microbiology and Biotechnology, 62(1), pp. 1–20. doi: https://doi.org/10.1007/s00253-003-1223-9.

Johnson, M. (2013) ‘Detergents: Triton X-100, Tween-20, and more’, Materials and Methods, 3, p. 163. doi: https://doi.org/10.13070/mm.en.3.163.

Kollnberger, S. D., Gutierrez-Castañeda, B., Foster-Cuevas, M., Corteyn, A. and Parkhouse, R. M. E. (2002) ‘Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody’, Journal of General Virology, 83(6), pp. 1331–1342. doi: https://doi.org/10.1099/0022-1317-83-6-1331.

Lau, B. Y. C. and Othman, A. (2019) ‘Evaluation of sodium deoxycholate as solubilization buffer for oil palm proteomics analysis’, PloS ONE, 14(8), p. e0221052. doi: https://doi.org/10.1371/journal.pone.0221052.

Ma, Y., Lee, C.-J. and Park, J.-S. (2020) ‘Strategies for optimizing the production of proteins and peptides with multiple disulfide bonds’, Antibiotics, 9(9), p. 541. doi: https://doi.org/10.3390/antibiotics9090541.

Mazur-Panasiuk, N., Żmudzki, J. and Woźniakowski, G. (2019) ‘African swine fever virus — persistence in different environmental conditions and the possibility of its indirect transmission’, Journal of Veterinary Research, 63(3), pp. 303–310. doi: https://doi.org/10.2478/jvetres-2019-0058.

OIE (World Organisation for Animal Health) (2021a) ‘Chapter 3.9.1. African Swine Fever (Infection with African Swine Fever Virus)’, in Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Bees). 8^th ed. [version adopted in May 2021]. Paris: OIE, pp. 1–18. Available at: https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.09.01_ASF.pdf.

OIE (World Organisation for Animal Health) (2021b) Global Situation of African Swine Fever. Report No 47: 2016–2020. Paris: OIE. Available at: https://www.oie.int/app/uploads/2021/03/report-47-global-situation-asf.pdf.

Oura, Ch. (2017) ‘Chapter 8 — Asfarviridae and Iridoviridae’, in MacLachlan, N. J. and Dubovi, E. J. (eds.) Fenner’s Veterinary Virology. 5^th ed. San Diego: Academic Press, pp. 175–188. doi: https://doi.org/10.1016/B978-0-12-800946-8.00008-8.

Pierce Biotechnology (2009) Tech Tip # 43. Protein Stability and Storage. Rockford, Il, USA: Thermo Fisher Scientific Inc.. Available at: https://tools.thermofisher.com/content/sfs/brochures/TR0043-Protein-storage.pdf.

Simpson, R. J. (2010) ‘Stabilization of proteins for storage’, Cold Spring Harbor Protocols, 2010(5), p. pdb.top79. doi: https://doi.org/10.1101/pdb.top79.

Singh, A., Upadhyay, V., Upadhyay, A. K., Singh, S. M. and Panda, A. K. (2015) ‘Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process’, Microbial Cell Factories, 14(1), p. 41. doi: https://doi.org/10.1186/s12934-015-0222-8.

SSUFSCP (State Service of Ukraine on Food Safety and Consumer Protection) (2021) African Swine Fever [Afrykanska chuma svynei]. Available at: https://www.asf.vet.ua. [in Ukrainian].

Tao, H., Liu, W., Simmons, B. N., Harris, H. K., Cox, T. C. and Massiah, M. A. (2010) ‘Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS’, BioTechniques, 48(1), pp. 61–64. doi: https://doi.org/10.2144/000113304.

Thomas, P. and Smart, T. G. (2005) ‘HEK293 cell line: A vehicle for the expression of recombinant proteins’, Journal of Pharmacological and Toxicological Methods, 51(3), pp. 187–200. doi: https://doi.org/10.1016/j.vascn.2004.08.014.

Yang, Z., Zhang, L., Zhang, Y., Zhang, T., Feng, Y., Lu, X., Lan, W., Wang, J., Wu, H., Cao, C. and Wang, X. (2011) ‘Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method’, PLoS ONE, 6(7), p. e22981. doi: https://doi.org/10.1371/journal.pone.0022981.

Zani, L., Forth, J. H., Forth, L., Nurmoja, I., Leidenberger, S., Henke, J., Carlson, J., Breidenstein, C., Viltrop, A., Höper, D., Sauter-Louis, C., Beer, M. and Blome, S. (2018) ‘Deletion at the 5’-end of Estonian ASFV strains associated with an attenuated phenotype’, Scientific Reports, 8(1), p. 6510. doi: https://doi.org/10.1038/s41598-018-24740-1.

Zhao, X., Li, G. and Liang, S. (2013) ‘Several affinity tags commonly used in chromatographic purification’, Journal of Analytical Methods in Chemistry, 2013, pp. 581093. doi: https://doi.org/10.1155/2013/581093.