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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

Nucleotide sequence of the gene coding the protein NP49-375 highlighting the rare codons.
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Figure 1: Nucleotide sequence of the gene coding the protein NP49-375 highlighting the rare codons.

Mentions: The recombinant expression plasmid, named pET-28a-np49-375, was assembled by subcloning the PCR product previously phosphorylated into the plasmid pUC18 (Thermo Scientific, USA) digested with the enzyme Sma I, obtaining the plasmid pUC-np49-375. Subsequently, the DNA segment coding the protein NP49-375 was removed from the plasmid pUC-np49-375 by digestion with the enzymes Nhe I and EcoR I (Promega, USA) and inserted into the prokaryotic expression vector pET-28a (Invitrogen, USA), previously digested with the same enzymes, to obtain the final construction. The plasmids pUC-np49-375 and pET-28a-np49-375 were sequenced (Macrogen, South Korea) and checked by a restriction assay using the restriction enzymes Nhe I and EcoR I to confirm the authenticity of the gene of interest. The E. coli strains BL21-CodonPlus® (DE3)-RIL (Stratagene, USA), BL21-CodonPlus® (DE3)-RP (Stratagene, USA) and Rosetta™ (DE3) (Novagen, Germany) were transformed with the plasmid pET-28a-np49-375 following the procedures of the instruction manual of BL21-CodonPlus® Competent Cells (Stratagene, USA). We performed the expression induction of the gene coding the protein NP49-375 following the instructions of the same manual. The E. coli strains were selected due to previous failure in the expression of the gene np49-375 using the E. coli strain BL-21 (DE3) as host and the existence of several rare codons in the nucleotide sequence of this gene, which could impair the protein translation process (Fig. 1).


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)

Nucleotide sequence of the gene coding the protein NP49-375 highlighting the rare codons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Nucleotide sequence of the gene coding the protein NP49-375 highlighting the rare codons.
Mentions: The recombinant expression plasmid, named pET-28a-np49-375, was assembled by subcloning the PCR product previously phosphorylated into the plasmid pUC18 (Thermo Scientific, USA) digested with the enzyme Sma I, obtaining the plasmid pUC-np49-375. Subsequently, the DNA segment coding the protein NP49-375 was removed from the plasmid pUC-np49-375 by digestion with the enzymes Nhe I and EcoR I (Promega, USA) and inserted into the prokaryotic expression vector pET-28a (Invitrogen, USA), previously digested with the same enzymes, to obtain the final construction. The plasmids pUC-np49-375 and pET-28a-np49-375 were sequenced (Macrogen, South Korea) and checked by a restriction assay using the restriction enzymes Nhe I and EcoR I to confirm the authenticity of the gene of interest. The E. coli strains BL21-CodonPlus® (DE3)-RIL (Stratagene, USA), BL21-CodonPlus® (DE3)-RP (Stratagene, USA) and Rosetta™ (DE3) (Novagen, Germany) were transformed with the plasmid pET-28a-np49-375 following the procedures of the instruction manual of BL21-CodonPlus® Competent Cells (Stratagene, USA). We performed the expression induction of the gene coding the protein NP49-375 following the instructions of the same manual. The E. coli strains were selected due to previous failure in the expression of the gene np49-375 using the E. coli strain BL-21 (DE3) as host and the existence of several rare codons in the nucleotide sequence of this gene, which could impair the protein translation process (Fig. 1).

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