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Examination of immunogenic properties of recombinant antigens based on p22 protein from African swine fever virus

Carlos Díaz
Carlos Díaz
, 
Jiří Salát
Jiří Salát
, 
Dagmar Břínek Kolařová
Dagmar Břínek Kolařová
, 
Vladimír Celer
Vladimír Celer
 and 
Ivo Frébort
Ivo Frébort
   | Aug 30, 2022
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Published Online: Aug 30, 2022
Page range: 297 - 304
Received: May 03, 2022
Accepted: Aug 10, 2022
DOI: https://doi.org/10.2478/jvetres-2022-0043
Keywords
© 2022 C. Díaz et al. published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Predicted protein structure of the p22 C terminal protein (amino acids 42–189) by the Phyre2 protein fold recognition server (15). The colours progress as in a rainbow from the N- to the C-terminus
Predicted protein structure of the p22 C terminal protein (amino acids 42–189) by the Phyre2 protein fold recognition server (15). The colours progress as in a rainbow from the N- to the C-terminus

Fig. 2

DNA construct design of p22 C terminal (p22Ct) (left) and heat-labile enterotoxin B subunit fused with p22 C terminal (LTB-p22Ct) (right) sequences, cloned into the pET28b(+) expression vector. The p22Ct gene is coloured ruby red, the LTB coding sequence is orange, the pink colour represents the coding sequence for a 10× His-tag, yellow sections are the origins of replication, pale green the kanamycin resistance gene KanR and blue the multicloning site (MCS)
DNA construct design of p22 C terminal (p22Ct) (left) and heat-labile enterotoxin B subunit fused with p22 C terminal (LTB-p22Ct) (right) sequences, cloned into the pET28b(+) expression vector. The p22Ct gene is coloured ruby red, the LTB coding sequence is orange, the pink colour represents the coding sequence for a 10× His-tag, yellow sections are the origins of replication, pale green the kanamycin resistance gene KanR and blue the multicloning site (MCS)

Fig. 3

Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of crude extracts from E. coli BL21 Star cells expressing p22 C terminal (p22Ct) and heat-labile enterotoxin B subunit fused with p22 C terminal (LTB-p22Ct). Lanes 1, 3, 5, and 7 show the 20 μL samples from non-induced cells, while lanes 2, 4, 6, and 8 represent the cells after induction with 0.2 mM isopropyl β-D-1-thiogalactopyranoside. Bands corresponding to the p22Ct (lanes 2 and 4) and LTB-p22Ct (lanes 6 and 8) proteins are shown in red boxes
Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of crude extracts from E. coli BL21 Star cells expressing p22 C terminal (p22Ct) and heat-labile enterotoxin B subunit fused with p22 C terminal (LTB-p22Ct). Lanes 1, 3, 5, and 7 show the 20 μL samples from non-induced cells, while lanes 2, 4, 6, and 8 represent the cells after induction with 0.2 mM isopropyl β-D-1-thiogalactopyranoside. Bands corresponding to the p22Ct (lanes 2 and 4) and LTB-p22Ct (lanes 6 and 8) proteins are shown in red boxes

Fig. 4

Expression and purification of p22 C terminal (p22Ct) (A–C) and heat-labile enterotoxin B subunit fused with p22 C terminal (LTB-p22Ct) (D–F) recombinant proteins in E. coli BL21 analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot. A – band 1: cells harbouring empty pET28b(+) as control (after induction); band 2: cells harbouring pET28b(+)-p22Ct before induction; band 3: soluble protein fraction; band 4: cells after induction (each sample 20 μL); band 5: washout fraction (50 mM imidazole) from nickel-nitrilotriacetic agarose (20 μL); bands 6–9: p22Ct eluted by 300 mM imidazole (samples of 5, 10, 4, and 0.5 μg, respectively); B– protein staining; C – Western blot with the anti-His-tag antibody of the purified p22Ct protein (11 μg); D – band 1: pET28b(+) cells after induction; band 2: pET28b(+)-LTB-p22Ct cells before induction; band 3: soluble protein fraction; band 4: cells after induction (each 20 μL); band 5: washout fraction; bands 6–9: eluted LTB-p22Ct protein (4, 3, 2 and 0 μg); E – protein staining; F – Western blot of purified LTB-p22Ct (16 μg); M – protein molecular mass marker
Expression and purification of p22 C terminal (p22Ct) (A–C) and heat-labile enterotoxin B subunit fused with p22 C terminal (LTB-p22Ct) (D–F) recombinant proteins in E. coli BL21 analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot. A – band 1: cells harbouring empty pET28b(+) as control (after induction); band 2: cells harbouring pET28b(+)-p22Ct before induction; band 3: soluble protein fraction; band 4: cells after induction (each sample 20 μL); band 5: washout fraction (50 mM imidazole) from nickel-nitrilotriacetic agarose (20 μL); bands 6–9: p22Ct eluted by 300 mM imidazole (samples of 5, 10, 4, and 0.5 μg, respectively); B– protein staining; C – Western blot with the anti-His-tag antibody of the purified p22Ct protein (11 μg); D – band 1: pET28b(+) cells after induction; band 2: pET28b(+)-LTB-p22Ct cells before induction; band 3: soluble protein fraction; band 4: cells after induction (each 20 μL); band 5: washout fraction; bands 6–9: eluted LTB-p22Ct protein (4, 3, 2 and 0 μg); E – protein staining; F – Western blot of purified LTB-p22Ct (16 μg); M – protein molecular mass marker

Fig. 5

Immunoblot assay of the purified p22 C terminal and heat-labile enterotoxin B subunit fused with p22 C terminal recombinant proteins with four different sera from African swine fever virus–infected pigs. The binding intensity was calculated as the intensity of the bands compared to the background over the whole lane of the immunoblot assay
Immunoblot assay of the purified p22 C terminal and heat-labile enterotoxin B subunit fused with p22 C terminal recombinant proteins with four different sera from African swine fever virus–infected pigs. The binding intensity was calculated as the intensity of the bands compared to the background over the whole lane of the immunoblot assay

Fig. 6

ELISA test results of sera samples taken from mice groups immunised with p22 C terminal and heat-labile enterotoxin B subunit fused with p22 C terminal
ELISA test results of sera samples taken from mice groups immunised with p22 C terminal and heat-labile enterotoxin B subunit fused with p22 C terminal

Fig. 7

Gel electrophoresis of p22 C terminal (13 μg) and heat-labile enterotoxin B subunit fused with-p22 C terminal (16 μg) samples under reducing (lanes 2 and 4) and non-reducing conditions without adding 2-mercaptoethanol or heating (lanes 1 and 3); M – protein molecular mass marker
Gel electrophoresis of p22 C terminal (13 μg) and heat-labile enterotoxin B subunit fused with-p22 C terminal (16 μg) samples under reducing (lanes 2 and 4) and non-reducing conditions without adding 2-mercaptoethanol or heating (lanes 1 and 3); M – protein molecular mass marker
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