Cloning and expression of African swine fever virus K205R and B602L genes inf. COLI
Abstract
A molecular cloning of African swine fever virus k205r and b602l genes in £ coli was carried out. The expression and purification conditions ensuring high yield of recombinant proteins were optimized. Dissolved recombinant proteins were purified by metal-chelate affinity chromatography using Ni-NTA-agarose («Qiagen»). Recombinant antigens are biologically safe, easier to prepare and ensure higher ELISA specificity. The purified protein yield from 100 ml off. coli culture was 1,5 mg for pK205R and 2 mg for pB602L.
About the Authors
A. V. Scherbakov
FGBI "ARRIAH", Vladimir
Russian Federation
Russian Federation
A. S. Yakovleva
FGBI "ARRIAH", Vladimir
Russian Federation
Russian Federation
A. M. Timina
FGBI "ARRIAH", Vladimir
Russian Federation
Russian Federation
M. R. Yakupov
FGBI "ARRIAH", Vladimir
Russian Federation
Russian Federation
References
1. MANIATIS T., Fritsch E., Sambrook D. Genetic engineering techniques. Molecular cloning. - M: Mir, 1984. -480 p.
2. African swine fever // OIE. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. - 2012. - Vol. 2, Chap. 2.8.1. - URL: http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/ (date of visit: 01.10.14).
3. African swine fever/J.M. Sanchez-Vizcaino, B. Straw, S. D'Allaire, W. Mengeling // Diseases of Swine / ed. B.E. Straw [et al.]. - 9th ed. - Ames, Iowa, 2006. - P. 291 -298.
4. African swine fever virus serodiagnosis: a general review with a focus on the analyses of African serum samples / C. Cubilos, S. Gomez, N. Moreno [et al.] //Virus Res. - 2013. - Vol. 173. - P. 159-167.
5. Antigenic properties and diagnostic potential of African swine fever virus protein pp62 expressed in insect cells/С. Gallardo, E. Blanco, J.M. Rodriguez [etal.]//J. Clin. Microbiol. - 2006. - Vol. 44. - P. 950-956.
6. Escribano J.M., Pastor M.J., Sanchez Vizcaino J.M. Antibodies to bovine serum albumin in swine sera: implications for false positive reactions in the serodiagnosis of African swine fever // Am. J. Vet. Res. - 1989.-Vol. 50. - P. 1118-1122.
7. High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents / J.M. Oviedo, F. Rodriguez, P. Gomez-Puertas [et al.] // J. Virol. Meth.- 1997,-Vol. 64,-P. 27-35.
8. Highly specific confirmatory western blot test of African swine fever virus antibody detection using the recombinant virus protein p54 / C. Alcaraz, F. Rodriguez, J. Oviedo [et al.] // J. Virol. Meth. - 1995. - Vol. 52. - P. 111 -119.
9. Optimization and validation of recombinant serological tests for African swine fever diagnosis based on detection of the p30 protein produced on Trichoplusia ni larvae / D.M. Perez-Filgueira, F. Gonzalez-Camacho, C. Gallardo [et al.] // J. Clin. Microbiol. - 2006.-Vol. 44. - P. 3114-3121.
10. Recombinant antigen targets for serodiagnosis of African swine fever / C. Gallardo, A.L. Reis, G. Kalema-Zikusoka [et al.] // Clin. Vaccine Immunol.- 2009,- Vol. 16. -P. 1012-1020.
11. Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae / M. Barderas, A. Wigdorovitz, F. Merelo [et al.] // J. Virol. Meth. -2000,-Vol. 89,-P. 129-136.
12. Systematic analysis of longitudinal serological responses of pigs infected experimentally with African swine fever virus / A.L. Reis, R.M. Parkhouse, A.R. Penedos [et al.] // J. Gen. Virol. - 2007. - Vol. 88. - P. 2426-2434.
Review
For citations:
Scherbakov A.V., Yakovleva A.S., Timina A.M., Yakupov M.R. Cloning and expression of African swine fever virus K205R and B602L genes inf. COLI. Veterinary Science Today. 2015;(2):31-35. (In Russ.)
Views:
530
Email this article 