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Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study.

Pose AG, Rodríguez ES, Méndez AC, Gómez JN, Redondo AV, Rodríguez ER, Ramos EM, Gutiérrez AÁ, Moltó MP, Roche DG, Ugalde YS, López AM - Open Vet J (2015)

Bottom Line: As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli.The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies.No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases.

View Article: PubMed Central - PubMed

Affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.

ABSTRACT
In this study we demonstrated that the vaccine candidate against avian influenza virus H5N1 based on the hemagglutinin H5 (HA) fused to the chicken CD154 (HACD) can also be used for differentiating infected from vaccinated animals (DIVA). As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli. After its purification by IMAC, the competence of the proteins NP49-375 and HACD as coating antigens in indirect ELISA assays were tested by using the sera of chickens immunized with the proteins HA and HACD and the reference sera from several avian influenza subtypes. Together with these sera, the sera from different species of birds and the sera of chickens infected with other avian viral diseases were analyzed by competition ELISA assays coated with the proteins NP49-375 and HACD. The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies. Also, we observed variable detection levels when the reference sera were analyzed in the ELISA plates coated with the protein NP49-375. Moreover, only the antibodies of the reference serum subtype H5 were detected in the ELISA plates coated with the protein HACD. The competition ELISA assays showed percentages of inhibition of 88-91% for the positives sera and less than 20% for the negative sera. We fixed the cut-off value of these assays at 25%. No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases. This study supported the fact that the ELISA assays using the proteins NP49-375 and HACD could be valuable tools for avian influenza surveillance and as a strategy of DIVA for counteracting the highly pathogenic avian influenza virus H5N1 outbreaks.

No MeSH data available.


Related in: MedlinePlus

Solubilization and purification of the protein NP49-375. (A) SDS-PAGE (12.5%) of the different fractions after the rupture. 1- MWM (New England Biolabs, USA). 2- Rupture pellet. 3- Rupture supernatant (B) Solubilization with GuHCl. 1- Pellet GuHCl 1M, 2- Pellet GuHCl 2M, 3- Pellet GuHCl 4M, 4- Pellet GuHCl 6M, 5- Supernatant GuHCl 1M, 6- Supernatant GuHCl 2M, 7- Supernatant GuHCl 4M, 8- Supernatant GuHCl 6M. SDS-PAGE (12.5%) (C) and Western blot (D) of the different stages in the purification process performed by IMAC. 1- MWM (New England Biolabs, USA), 2- Initial sample, 3- Non-attached proteins, 4- Wash 20 mM Imidazole, 5- Elution 100 mM Imidazole.
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Figure 4: Solubilization and purification of the protein NP49-375. (A) SDS-PAGE (12.5%) of the different fractions after the rupture. 1- MWM (New England Biolabs, USA). 2- Rupture pellet. 3- Rupture supernatant (B) Solubilization with GuHCl. 1- Pellet GuHCl 1M, 2- Pellet GuHCl 2M, 3- Pellet GuHCl 4M, 4- Pellet GuHCl 6M, 5- Supernatant GuHCl 1M, 6- Supernatant GuHCl 2M, 7- Supernatant GuHCl 4M, 8- Supernatant GuHCl 6M. SDS-PAGE (12.5%) (C) and Western blot (D) of the different stages in the purification process performed by IMAC. 1- MWM (New England Biolabs, USA), 2- Initial sample, 3- Non-attached proteins, 4- Wash 20 mM Imidazole, 5- Elution 100 mM Imidazole.

Mentions: After the BL21-CodonPlus® (DE3)-RIL strain transformed with the plasmid pET-28a-np49-375 was grown at a favorable optical density, the bacterial culture was harvested and sonicated. The protein NP49-375 was obtained as insoluble inclusion bodies in the lysate (Fig. 4A). It was solubilized using different concentrations of GuHCl (Fig. 4B). The rise of the GuHCl concentration at 1 M, 2 M, 4 M and 6 M increased the solubilization properties of the protein NP49-375 and provoked its gradual transition from the lysate to the supernatant.


Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study.

Pose AG, Rodríguez ES, Méndez AC, Gómez JN, Redondo AV, Rodríguez ER, Ramos EM, Gutiérrez AÁ, Moltó MP, Roche DG, Ugalde YS, López AM - Open Vet J (2015)

Solubilization and purification of the protein NP49-375. (A) SDS-PAGE (12.5%) of the different fractions after the rupture. 1- MWM (New England Biolabs, USA). 2- Rupture pellet. 3- Rupture supernatant (B) Solubilization with GuHCl. 1- Pellet GuHCl 1M, 2- Pellet GuHCl 2M, 3- Pellet GuHCl 4M, 4- Pellet GuHCl 6M, 5- Supernatant GuHCl 1M, 6- Supernatant GuHCl 2M, 7- Supernatant GuHCl 4M, 8- Supernatant GuHCl 6M. SDS-PAGE (12.5%) (C) and Western blot (D) of the different stages in the purification process performed by IMAC. 1- MWM (New England Biolabs, USA), 2- Initial sample, 3- Non-attached proteins, 4- Wash 20 mM Imidazole, 5- Elution 100 mM Imidazole.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663798&req=5

Figure 4: Solubilization and purification of the protein NP49-375. (A) SDS-PAGE (12.5%) of the different fractions after the rupture. 1- MWM (New England Biolabs, USA). 2- Rupture pellet. 3- Rupture supernatant (B) Solubilization with GuHCl. 1- Pellet GuHCl 1M, 2- Pellet GuHCl 2M, 3- Pellet GuHCl 4M, 4- Pellet GuHCl 6M, 5- Supernatant GuHCl 1M, 6- Supernatant GuHCl 2M, 7- Supernatant GuHCl 4M, 8- Supernatant GuHCl 6M. SDS-PAGE (12.5%) (C) and Western blot (D) of the different stages in the purification process performed by IMAC. 1- MWM (New England Biolabs, USA), 2- Initial sample, 3- Non-attached proteins, 4- Wash 20 mM Imidazole, 5- Elution 100 mM Imidazole.
Mentions: After the BL21-CodonPlus® (DE3)-RIL strain transformed with the plasmid pET-28a-np49-375 was grown at a favorable optical density, the bacterial culture was harvested and sonicated. The protein NP49-375 was obtained as insoluble inclusion bodies in the lysate (Fig. 4A). It was solubilized using different concentrations of GuHCl (Fig. 4B). The rise of the GuHCl concentration at 1 M, 2 M, 4 M and 6 M increased the solubilization properties of the protein NP49-375 and provoked its gradual transition from the lysate to the supernatant.

Bottom Line: As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli.The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies.No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases.

View Article: PubMed Central - PubMed

Affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.

ABSTRACT
In this study we demonstrated that the vaccine candidate against avian influenza virus H5N1 based on the hemagglutinin H5 (HA) fused to the chicken CD154 (HACD) can also be used for differentiating infected from vaccinated animals (DIVA). As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli. After its purification by IMAC, the competence of the proteins NP49-375 and HACD as coating antigens in indirect ELISA assays were tested by using the sera of chickens immunized with the proteins HA and HACD and the reference sera from several avian influenza subtypes. Together with these sera, the sera from different species of birds and the sera of chickens infected with other avian viral diseases were analyzed by competition ELISA assays coated with the proteins NP49-375 and HACD. The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies. Also, we observed variable detection levels when the reference sera were analyzed in the ELISA plates coated with the protein NP49-375. Moreover, only the antibodies of the reference serum subtype H5 were detected in the ELISA plates coated with the protein HACD. The competition ELISA assays showed percentages of inhibition of 88-91% for the positives sera and less than 20% for the negative sera. We fixed the cut-off value of these assays at 25%. No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases. This study supported the fact that the ELISA assays using the proteins NP49-375 and HACD could be valuable tools for avian influenza surveillance and as a strategy of DIVA for counteracting the highly pathogenic avian influenza virus H5N1 outbreaks.

No MeSH data available.


Related in: MedlinePlus