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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

Isolation of a segment of the gene np and construction of the expression vector. (A) Cloning representation of a gene coding for a segment of the protein NP from avian influenza virus subtype H7N1 comprising the aminoacids 49-375 (np49-375) in the plasmid pUC-18 and in the expression vector pET-28a. (B) Isolation of the gene np49-375 by PCR. 1- Molecular weight marker (MWM) (pAdEasy digested with the enzyme Apa I), 2- DNA segment corresponding to the gene np49-375, 3- PCR reaction with primers and without template. (C) Electrophoresis in agarose gel (1%) of the restriction analysis for the plasmid pUC-np49-375. 1- MWM, 2- Plasmid pUC-np49-375 undigested, 3- Plasmid pUC18 digested with the enzymes Nhe I/EcoR I, 4- Plasmid pUC-np49-375 digested with the enzymes Nhe I/EcoR I. (D) Electrophoresis in agarose gel (1%) of the restriction analysis for the plasmid pET-28a-np49-375. 1- MWM, 2- Plasmid pET-28a-np49-375 undigested, 3- Plasmid pET-28a digested with the enzymes Nhe I/EcoR I, 4- Plasmid pET-28a-np49-375 digested with the enzymes Nhe I/EcoR I.
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Figure 2: Isolation of a segment of the gene np and construction of the expression vector. (A) Cloning representation of a gene coding for a segment of the protein NP from avian influenza virus subtype H7N1 comprising the aminoacids 49-375 (np49-375) in the plasmid pUC-18 and in the expression vector pET-28a. (B) Isolation of the gene np49-375 by PCR. 1- Molecular weight marker (MWM) (pAdEasy digested with the enzyme Apa I), 2- DNA segment corresponding to the gene np49-375, 3- PCR reaction with primers and without template. (C) Electrophoresis in agarose gel (1%) of the restriction analysis for the plasmid pUC-np49-375. 1- MWM, 2- Plasmid pUC-np49-375 undigested, 3- Plasmid pUC18 digested with the enzymes Nhe I/EcoR I, 4- Plasmid pUC-np49-375 digested with the enzymes Nhe I/EcoR I. (D) Electrophoresis in agarose gel (1%) of the restriction analysis for the plasmid pET-28a-np49-375. 1- MWM, 2- Plasmid pET-28a-np49-375 undigested, 3- Plasmid pET-28a digested with the enzymes Nhe I/EcoR I, 4- Plasmid pET-28a-np49-375 digested with the enzymes Nhe I/EcoR I.

Mentions: The Figure 2A shows the schematic representation of the cloning steps followed to generate the expression vector coding the protein NP49-375. After obtaining the complementary DNA from the viral RNA of a specific avian influenza viral strain, the gene coding the protein NP49-375 was amplified by PCR. The electrophoresis in agarose gel (0.8%) showed a DNA segment of 1002 base pair corresponding to the size of the gene of interest (Fig. 2B). Next, the gene was subcloned into the plasmid pUC-18 in order to facilitate its manipulation and finally cloned into the expression vector pET-28a obtaining the plasmids pUC-np49-375 and pET-28a-np49-375, respectively. To corroborate the authenticity of the gene of interest, the plasmids were submitted to a restriction assay using the enzymes Nhe I and EcoR I after sequencing. Both plasmids showed the expected pattern of DNA segments (Fig. 2C and D).


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)

Isolation of a segment of the gene np and construction of the expression vector. (A) Cloning representation of a gene coding for a segment of the protein NP from avian influenza virus subtype H7N1 comprising the aminoacids 49-375 (np49-375) in the plasmid pUC-18 and in the expression vector pET-28a. (B) Isolation of the gene np49-375 by PCR. 1- Molecular weight marker (MWM) (pAdEasy digested with the enzyme Apa I), 2- DNA segment corresponding to the gene np49-375, 3- PCR reaction with primers and without template. (C) Electrophoresis in agarose gel (1%) of the restriction analysis for the plasmid pUC-np49-375. 1- MWM, 2- Plasmid pUC-np49-375 undigested, 3- Plasmid pUC18 digested with the enzymes Nhe I/EcoR I, 4- Plasmid pUC-np49-375 digested with the enzymes Nhe I/EcoR I. (D) Electrophoresis in agarose gel (1%) of the restriction analysis for the plasmid pET-28a-np49-375. 1- MWM, 2- Plasmid pET-28a-np49-375 undigested, 3- Plasmid pET-28a digested with the enzymes Nhe I/EcoR I, 4- Plasmid pET-28a-np49-375 digested with the enzymes Nhe I/EcoR I.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Isolation of a segment of the gene np and construction of the expression vector. (A) Cloning representation of a gene coding for a segment of the protein NP from avian influenza virus subtype H7N1 comprising the aminoacids 49-375 (np49-375) in the plasmid pUC-18 and in the expression vector pET-28a. (B) Isolation of the gene np49-375 by PCR. 1- Molecular weight marker (MWM) (pAdEasy digested with the enzyme Apa I), 2- DNA segment corresponding to the gene np49-375, 3- PCR reaction with primers and without template. (C) Electrophoresis in agarose gel (1%) of the restriction analysis for the plasmid pUC-np49-375. 1- MWM, 2- Plasmid pUC-np49-375 undigested, 3- Plasmid pUC18 digested with the enzymes Nhe I/EcoR I, 4- Plasmid pUC-np49-375 digested with the enzymes Nhe I/EcoR I. (D) Electrophoresis in agarose gel (1%) of the restriction analysis for the plasmid pET-28a-np49-375. 1- MWM, 2- Plasmid pET-28a-np49-375 undigested, 3- Plasmid pET-28a digested with the enzymes Nhe I/EcoR I, 4- Plasmid pET-28a-np49-375 digested with the enzymes Nhe I/EcoR I.
Mentions: The Figure 2A shows the schematic representation of the cloning steps followed to generate the expression vector coding the protein NP49-375. After obtaining the complementary DNA from the viral RNA of a specific avian influenza viral strain, the gene coding the protein NP49-375 was amplified by PCR. The electrophoresis in agarose gel (0.8%) showed a DNA segment of 1002 base pair corresponding to the size of the gene of interest (Fig. 2B). Next, the gene was subcloned into the plasmid pUC-18 in order to facilitate its manipulation and finally cloned into the expression vector pET-28a obtaining the plasmids pUC-np49-375 and pET-28a-np49-375, respectively. To corroborate the authenticity of the gene of interest, the plasmids were submitted to a restriction assay using the enzymes Nhe I and EcoR I after sequencing. Both plasmids showed the expected pattern of DNA segments (Fig. 2C and D).

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