Prokaryotic expression, purification and antigenicity analysis of African swine fever virus pK205R protein

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African swine fever is an acute, febrile and highly virulent porcine disease causing serious economic losses worldwide. The pK205R protein of the African swine fever virus (ASFV) is largely expressed in the early stages of infection, which has given the K205R gene extensive attention. In this study, the ASFV K205R was cloned and expressed in Escherichia coli BL21 (DE3). Expression of histidine-tagged pK205R with a molecular mass of 44 kDa was determined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Optimisation of culture conditions allowed induction of the recombinant protein with 0.4 mM Isopropyl β-D-thiogalactoside (IPTG) at 37°C for 2 h. The protein existed in cellular supernatant and was purified using a Ni-NTA resin column. The purified protein was used to immunize rabbits four times to enable the production of polyclonal antibodies, and the antiserum titre was detected by ELISA. The results showed that the purified pK205R can react with ASFV positive serum specifically by Western blotting. The pK205R had high antigenicity, which indicated that pK205R could be used as an antigen for detection of ASFV-specific antibodies in ELISA testing, and the recombinant protein could contribute to further research of the action and structure of pK205R.

Atuhaire DK, Afayoa M, Ochwo S, Mwesigwa S, Mwiine FN, Okuni JB, Olaho-Mukani W, Ojok L (2013) Prevalence of African swine fever virus in apparently healthy domestic pigs in Uganda. BMC Vet Res 9: 263.

Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22: 1399-1408.

Burrage TG (2013) African swine fever virus infection in Ornithodoros ticks. Virus Res 173: 131-139.

Cubillos C, Gomez-Sebastian S, Moreno N, Nunez MC, Mulumba-Mfumu LK, Quembo CJ, Heath L, Etter EM, Jori F, Escribano JM, Blanco E (2013) African swine fever virus serodiagnosis: a general review with a focus on the analyses of African serum samples. Virus Res 173: 159-167.

de Glanville WA, Vial L, Costard S, Wieland B, Pfeiffer DU (2014) Spatial multi-criteria decision analysis to predict suitability for African swine fever endemicity in Africa. BMC Vet Res 10: 9.

Diaz AV, Netherton CL, Dixon LK, Wilson AJ (2012) African swine fever virus strain Georgia 2007/1 in Ornithodoros erraticus ticks. Emerg Infect Dis 18: 1026-1028.

Gomez-Villamandos JC, Bautista MJ, Sanchez-Cordon PJ, Carrasco L (2013) Pathology of African swine fever: the role of monocyte-macrophage. Virus Res 173: 140-149.

Gutierrez-Castaneda B, Reis AL, Corteyn A, Parkhouse RM, Kollnberger S (2008) Expression, cellular localization and antibody responses of the African swine fever virus genes B602L and K205R. Arch Virol 153: 2303-2306.

Hu Z, Chen Z, Huang N, Teng X, Zhang J, Wang Z, Wei X, Qin K, Liu X, Wu X, Tang H, Zhu X, Cui K, Li J (2015) Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies. Protein Expr Purif 107: 76-82.

Kirk WR (2014) Thermodynamics of imidazole-ligand binding to Ni-nitrilotriacetate in solution and covalently attached to agarose beads: imidazole, his-6 (his-tag) peptide and a new bis-imidazolo-dithiane. Protein Expr Purif 95: 1-7.

Liu D, Si B, Li C, Mi Z, An X, Qin C, Liu W, Tong Y (2011) Prokaryotic expression and purification of HA1 and HA2 polypeptides for serological analysis of the 2009 pandemic H1N1 influenza virus. J Virol Methods 172: 16-21.

Montgomery RE (1921) On a form of swine fever occurring in British East Africa (Kenya Colony). Journal of Comparative Pathology 34: 159-191.

Oganesyan AS, Petrova ON, Korennoy FI, Bardina NS, Gogin AE, Dudnikov SA (2013) African swine fever in the Russian Federation: spatio-temporal analysis and epidemiological overview. Virus Res 173: 204-211.

Pacheco B, Crombet L, Loppnau P, Cossar D (2012) A screening strategy for heterologous protein expression in Escherichia coli with the highest return of investment. Protein Expr Purif 81: 33-41.

Paton DJ, Taylor G (2011) Developing vaccines against foot-and-mouth disease and some other exotic viral diseases of livestock. Philos Trans R Soc Lond B Biol Sci 366: 2774-2781.

Ronish B, Hakhverdyan M, Stahl K, Gallardo C, Fernandez- Pinero J, Belak S, Leblanc N, Wangh L (2011) Design and verification of a highly reliable Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus. J Virol Methods 172: 8-15.

Sampoli Benitez B, Barbati ZR, Arora K, Bogdanovic J, Schlick T (2013) How DNA polymerase X preferentially accommodates incoming dATP opposite 8-oxoguanine on the template. Biophys J 105: 2559-2568.

Sanchez EG, Quintas A, Nogal M, Castello A, Revilla Y (2013) African swine fever virus controls the host transcription and cellular machinery of protein synthesis. Virus Res 173: 58-75.

Shenyang G, Enhui Z, Baoxian L, Xinyuan Q, Lijie T, Junwei G, Yijing L (2007) High-level prokaryotic expression of envelope exterior of membrane protein of porcine epidemic diarrhea virus. Vet Microbiol 123: 187-193.

Takamatsu HH, Denyer MS, Lacasta A, Stirling CM, Argilaguet JM, Netherton CL, Oura CA, Martins C, Rodriguez F (2013) Cellular immunity in ASFV responses. Virus Res 173: 110-121.

Uttenthal A, Braae UC, Ngowi HA, Rasmussen TB, Nielsen J, Johansen MV (2013) ASFV in Tanzania: asymptomatic pigs harbor virus of molecular similarity to Georgia 2007. Vet Microbiol 165: 173-176.

Wieland B, Dhollander S, Salman M, Koenen F (2011) Qualitative risk assessment in a data-scarce environment: a model to assess the impact of control measures on spread of African Swine Fever. Prev Vet Med 99: 4-14.

Zhang Y, Taiming L, Liu J (2003) Low temperature and glucose enhanced T7 RNA polymerase-based plasmid stability for increasing expression of glucagon-like peptide-2 in Escherichia coli. Protein Expr Purif 29: 132-139

Polish Journal of Veterinary Sciences

The Journal of Committee of Veterinary Sciences of Polish Academy of Sciences and University of Warmia and Mazury in Olsztyn

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