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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

Expression of the gene coding the protein NP49-375 in different E. coli strains. SDS-PAGE (12.5%) and Western blot of the samples in the non-induced (A) or induced (B) stages on different E. coli strains. 1- MWM (Bio-Rad, USA), 2- Untransformed BL-21-Codon Plus® (DE3)-RIL, 3- Untransformed BL-21-Codon Plus® (DE3)-RP, 4- Untransformed Rosetta™ (DE3), 5- BL-21-Codon Plus® (DE3)-RIL transformed with pET-28a-np49-375, 6- BL-21-Codon Plus® (DE3)-RP transformed with pET-28a-np49-375, 7- Rosetta™ (DE3) transformed with pET-28a-np49-375. Immuno identification was performed with a monoclonal antibody against the six histidine residues (Sigma, USA). Arrow heads indicate the protein NP49-375.
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Figure 3: Expression of the gene coding the protein NP49-375 in different E. coli strains. SDS-PAGE (12.5%) and Western blot of the samples in the non-induced (A) or induced (B) stages on different E. coli strains. 1- MWM (Bio-Rad, USA), 2- Untransformed BL-21-Codon Plus® (DE3)-RIL, 3- Untransformed BL-21-Codon Plus® (DE3)-RP, 4- Untransformed Rosetta™ (DE3), 5- BL-21-Codon Plus® (DE3)-RIL transformed with pET-28a-np49-375, 6- BL-21-Codon Plus® (DE3)-RP transformed with pET-28a-np49-375, 7- Rosetta™ (DE3) transformed with pET-28a-np49-375. Immuno identification was performed with a monoclonal antibody against the six histidine residues (Sigma, USA). Arrow heads indicate the protein NP49-375.

Mentions: The expression of the gene np49-375 was performed in three different E. coli strains carrying tRNAs for rare codons by transforming them with the plasmid pET-28a-np49-375. The SDS-PAGE and Western blot assays showed that under repressive conditions, the gene of interest was not expressed (Fig. 3A). However, after induction we observed a band of protein at about 37 kDa in the E. coli strains BL21-CodonPlus® (DE3)-RIL, BL21-CodonPlus® (DE3)-RP and Rosetta™ (DE3), previously transformed with the plasmid pET-28a-np49-375 (Fig. 3B). This protein size corresponded to the one predicted for the protein NP49-375. It was not observed in the induced stage of non-transformed E. coli strains. For the final production of the protein NP49-375, the BL21-CodonPlus® (DE3)-RIL strain was selected.


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)

Expression of the gene coding the protein NP49-375 in different E. coli strains. SDS-PAGE (12.5%) and Western blot of the samples in the non-induced (A) or induced (B) stages on different E. coli strains. 1- MWM (Bio-Rad, USA), 2- Untransformed BL-21-Codon Plus® (DE3)-RIL, 3- Untransformed BL-21-Codon Plus® (DE3)-RP, 4- Untransformed Rosetta™ (DE3), 5- BL-21-Codon Plus® (DE3)-RIL transformed with pET-28a-np49-375, 6- BL-21-Codon Plus® (DE3)-RP transformed with pET-28a-np49-375, 7- Rosetta™ (DE3) transformed with pET-28a-np49-375. Immuno identification was performed with a monoclonal antibody against the six histidine residues (Sigma, USA). Arrow heads indicate the protein NP49-375.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Expression of the gene coding the protein NP49-375 in different E. coli strains. SDS-PAGE (12.5%) and Western blot of the samples in the non-induced (A) or induced (B) stages on different E. coli strains. 1- MWM (Bio-Rad, USA), 2- Untransformed BL-21-Codon Plus® (DE3)-RIL, 3- Untransformed BL-21-Codon Plus® (DE3)-RP, 4- Untransformed Rosetta™ (DE3), 5- BL-21-Codon Plus® (DE3)-RIL transformed with pET-28a-np49-375, 6- BL-21-Codon Plus® (DE3)-RP transformed with pET-28a-np49-375, 7- Rosetta™ (DE3) transformed with pET-28a-np49-375. Immuno identification was performed with a monoclonal antibody against the six histidine residues (Sigma, USA). Arrow heads indicate the protein NP49-375.
Mentions: The expression of the gene np49-375 was performed in three different E. coli strains carrying tRNAs for rare codons by transforming them with the plasmid pET-28a-np49-375. The SDS-PAGE and Western blot assays showed that under repressive conditions, the gene of interest was not expressed (Fig. 3A). However, after induction we observed a band of protein at about 37 kDa in the E. coli strains BL21-CodonPlus® (DE3)-RIL, BL21-CodonPlus® (DE3)-RP and Rosetta™ (DE3), previously transformed with the plasmid pET-28a-np49-375 (Fig. 3B). This protein size corresponded to the one predicted for the protein NP49-375. It was not observed in the induced stage of non-transformed E. coli strains. For the final production of the protein NP49-375, the BL21-CodonPlus® (DE3)-RIL strain was selected.

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