Cloning, expression and purification of the influenza A (H9N2) virus M2e antigen and truncated Mycobacterium tuberculosis HSP70 as a fusion protein in Pichia pastoris.
Identifieur interne : 000E97 ( Main/Exploration ); précédent : 000E96; suivant : 000E98Cloning, expression and purification of the influenza A (H9N2) virus M2e antigen and truncated Mycobacterium tuberculosis HSP70 as a fusion protein in Pichia pastoris.
Auteurs : Seyyed Mahmoud Ebrahimi [Iran] ; Majid Tebianian ; Hadi Toghyani ; Arash Memarnejadian ; Hamid Reza AttaranSource :
- Protein expression and purification [ 1096-0279 ] ; 2010.
Descripteurs français
- KwdFr :
- Animaux, Antigènes viraux (génétique), Antigènes viraux (immunologie), Antigènes viraux (métabolisme), Clonage moléculaire, Lapins, Mycobacterium tuberculosis (génétique), Pichia (génétique), Pichia (métabolisme), Protéines de fusion recombinantes (immunologie), Protéines de fusion recombinantes (isolement et purification), Protéines de fusion recombinantes (métabolisme), Protéines de la matrice virale (génétique), Protéines de la matrice virale (immunologie), Protéines de la matrice virale (métabolisme), Protéines du choc thermique HSP70 (génétique), Protéines du choc thermique HSP70 (métabolisme), Protéines recombinantes (métabolisme), Sous-type H9N2 du virus de la grippe A (métabolisme).
- MESH :
- génétique : Antigènes viraux, Mycobacterium tuberculosis, Pichia, Protéines de la matrice virale, Protéines du choc thermique HSP70.
- immunologie : Antigènes viraux, Protéines de fusion recombinantes, Protéines de la matrice virale.
- isolement et purification : Protéines de fusion recombinantes.
- métabolisme : Antigènes viraux, Pichia, Protéines de fusion recombinantes, Protéines de la matrice virale, Protéines du choc thermique HSP70, Protéines recombinantes, Sous-type H9N2 du virus de la grippe A.
- Animaux, Clonage moléculaire, Lapins.
English descriptors
- KwdEn :
- Animals, Antigens, Viral (genetics), Antigens, Viral (immunology), Antigens, Viral (metabolism), Cloning, Molecular, HSP70 Heat-Shock Proteins (genetics), HSP70 Heat-Shock Proteins (metabolism), Influenza A Virus, H9N2 Subtype (metabolism), Mycobacterium tuberculosis (genetics), Pichia (genetics), Pichia (metabolism), Rabbits, Recombinant Fusion Proteins (immunology), Recombinant Fusion Proteins (isolation & purification), Recombinant Fusion Proteins (metabolism), Recombinant Proteins (metabolism), Viral Matrix Proteins (genetics), Viral Matrix Proteins (immunology), Viral Matrix Proteins (metabolism).
- MESH :
- chemical , genetics : Antigens, Viral, HSP70 Heat-Shock Proteins, Viral Matrix Proteins.
- chemical , immunology : Antigens, Viral, Recombinant Fusion Proteins, Viral Matrix Proteins.
- chemical , isolation & purification : Recombinant Fusion Proteins.
- chemical , metabolism : Antigens, Viral, HSP70 Heat-Shock Proteins, Recombinant Fusion Proteins, Recombinant Proteins, Viral Matrix Proteins.
- genetics : Mycobacterium tuberculosis, Pichia.
- metabolism : Influenza A Virus, H9N2 Subtype, Pichia.
- Animals, Cloning, Molecular, Rabbits.
Abstract
Due to its conservation, the extracellular domain of the influenza A M2 protein (M2e) has the potential for being applied as a recombinant vaccine candidate against a wide range of strains, though its immunogenicity may need to be improved. The occurrence of several post-translational modifications within the structure of M2 protein may affect its immunopotency for the induction of humoral immune response. Herein, to construct a recombinant M2e-based vaccine candidate with the appropriate structural conformation and immunogenicity the corresponding nucleotide sequence from an H9N2 influenza strain was fused to the N-terminus of the truncated Mycobacterium tuberculosis HSP70(359-610), as a potent adjuvant, and following its cloning into the pPICZ alpha A plasmid the fusion gene was expressed in Pichia pastoris KM71H yeast. The secreted protein was then easily purified from the culture media, based on the presence of polyhistidine tag and used for the production of rabbit polyclonal antisera. This raised antisera could recognize the native M2e protein on the surface of H9N2 influenza virus-infected MDCK cells at a comparable level with the commercial H2N2-specific anti-M2 antibody, which was evidenced with immunofluorescence and cell-ELISA assays. These results not only re-emphasized on the conservancy of the M2e antigen, but also pointed towards the applicability of the M2e-HSP70(359-610) fusion protein for the induction of specific antibodies capable of binding to the native M2e antigen on the infected cells. Collectively, this study implied that purified M2e-HSP70(359-610) represents a promising vaccine candidate; however, its in vivo potency for the induction of protection remains to be evaluated.
Url:
DOI: 10.1016/j.pep.2009.11.001
PubMed: 19897044
Affiliations:
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Le document en format XML
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Box 31975/148, Karaj, Tehran, Iran. smebrahimi@shirazu.ac.ir</nlm:affiliation><country xml:lang="fr">Iran</country><wicri:regionArea>Department of Biotechnology, Razi Vaccine and Serum Research Institute (RVSRI), P.O. Box 31975/148, Karaj, Tehran</wicri:regionArea><wicri:noRegion>Tehran</wicri:noRegion></affiliation></author><author><name sortKey="Tebianian, Majid" sort="Tebianian, Majid" uniqKey="Tebianian M" first="Majid" last="Tebianian">Majid Tebianian</name></author><author><name sortKey="Toghyani, Hadi" sort="Toghyani, Hadi" uniqKey="Toghyani H" first="Hadi" last="Toghyani">Hadi Toghyani</name></author><author><name sortKey="Memarnejadian, Arash" sort="Memarnejadian, Arash" uniqKey="Memarnejadian A" first="Arash" last="Memarnejadian">Arash Memarnejadian</name></author><author><name sortKey="Attaran, Hamid Reza" sort="Attaran, Hamid Reza" uniqKey="Attaran H" first="Hamid Reza" last="Attaran">Hamid Reza Attaran</name></author></analytic><series><title level="j">Protein expression and purification</title><idno type="eISSN">1096-0279</idno><imprint><date when="2010" type="published">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Antigens, Viral (genetics)</term><term>Antigens, Viral (immunology)</term><term>Antigens, Viral (metabolism)</term><term>Cloning, Molecular</term><term>HSP70 Heat-Shock Proteins (genetics)</term><term>HSP70 Heat-Shock Proteins (metabolism)</term><term>Influenza A Virus, H9N2 Subtype (metabolism)</term><term>Mycobacterium tuberculosis (genetics)</term><term>Pichia (genetics)</term><term>Pichia (metabolism)</term><term>Rabbits</term><term>Recombinant Fusion Proteins (immunology)</term><term>Recombinant Fusion Proteins (isolation & purification)</term><term>Recombinant Fusion Proteins (metabolism)</term><term>Recombinant Proteins (metabolism)</term><term>Viral Matrix Proteins (genetics)</term><term>Viral Matrix Proteins (immunology)</term><term>Viral Matrix Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Antigènes viraux (génétique)</term><term>Antigènes viraux (immunologie)</term><term>Antigènes viraux (métabolisme)</term><term>Clonage moléculaire</term><term>Lapins</term><term>Mycobacterium tuberculosis (génétique)</term><term>Pichia (génétique)</term><term>Pichia (métabolisme)</term><term>Protéines de fusion recombinantes (immunologie)</term><term>Protéines de fusion recombinantes (isolement et purification)</term><term>Protéines de fusion recombinantes (métabolisme)</term><term>Protéines de la matrice virale (génétique)</term><term>Protéines de la matrice virale (immunologie)</term><term>Protéines de la matrice virale (métabolisme)</term><term>Protéines du choc thermique HSP70 (génétique)</term><term>Protéines du choc thermique HSP70 (métabolisme)</term><term>Protéines recombinantes (métabolisme)</term><term>Sous-type H9N2 du virus de la grippe A (métabolisme)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Antigens, Viral</term><term>HSP70 Heat-Shock Proteins</term><term>Viral Matrix Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Antigens, Viral</term><term>Recombinant Fusion Proteins</term><term>Viral Matrix Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Recombinant Fusion Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Antigens, Viral</term><term>HSP70 Heat-Shock Proteins</term><term>Recombinant Fusion Proteins</term><term>Recombinant Proteins</term><term>Viral Matrix Proteins</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Mycobacterium tuberculosis</term><term>Pichia</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Antigènes viraux</term><term>Mycobacterium tuberculosis</term><term>Pichia</term><term>Protéines de la matrice virale</term><term>Protéines du choc thermique HSP70</term></keywords><keywords scheme="MESH" qualifier="immunologie" xml:lang="fr"><term>Antigènes viraux</term><term>Protéines de fusion recombinantes</term><term>Protéines de la matrice virale</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Protéines de fusion recombinantes</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Influenza A Virus, H9N2 Subtype</term><term>Pichia</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Antigènes viraux</term><term>Pichia</term><term>Protéines de fusion recombinantes</term><term>Protéines de la matrice virale</term><term>Protéines du choc thermique HSP70</term><term>Protéines recombinantes</term><term>Sous-type H9N2 du virus de la grippe A</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Cloning, Molecular</term><term>Rabbits</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Clonage moléculaire</term><term>Lapins</term></keywords><keywords scheme="mix" xml:lang="it"><term>HSP70</term><term>Influenza A virus</term><term>M2e</term><term>Pichia pastoris</term><term>Vaccine</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Due to its conservation, the extracellular domain of the influenza A M2 protein (M2e) has the potential for being applied as a recombinant vaccine candidate against a wide range of strains, though its immunogenicity may need to be improved. The occurrence of several post-translational modifications within the structure of M2 protein may affect its immunopotency for the induction of humoral immune response. Herein, to construct a recombinant M2e-based vaccine candidate with the appropriate structural conformation and immunogenicity the corresponding nucleotide sequence from an H9N2 influenza strain was fused to the N-terminus of the truncated Mycobacterium tuberculosis HSP70(359-610), as a potent adjuvant, and following its cloning into the pPICZ alpha A plasmid the fusion gene was expressed in Pichia pastoris KM71H yeast. The secreted protein was then easily purified from the culture media, based on the presence of polyhistidine tag and used for the production of rabbit polyclonal antisera. This raised antisera could recognize the native M2e protein on the surface of H9N2 influenza virus-infected MDCK cells at a comparable level with the commercial H2N2-specific anti-M2 antibody, which was evidenced with immunofluorescence and cell-ELISA assays. These results not only re-emphasized on the conservancy of the M2e antigen, but also pointed towards the applicability of the M2e-HSP70(359-610) fusion protein for the induction of specific antibodies capable of binding to the native M2e antigen on the infected cells. Collectively, this study implied that purified M2e-HSP70(359-610) represents a promising vaccine candidate; however, its in vivo potency for the induction of protection remains to be evaluated.</div></front></TEI><affiliations><list><country><li>Iran</li></country></list><tree><noCountry><name sortKey="Attaran, Hamid Reza" sort="Attaran, Hamid Reza" uniqKey="Attaran H" first="Hamid Reza" last="Attaran">Hamid Reza Attaran</name><name sortKey="Memarnejadian, Arash" sort="Memarnejadian, Arash" uniqKey="Memarnejadian A" first="Arash" last="Memarnejadian">Arash Memarnejadian</name><name sortKey="Tebianian, Majid" sort="Tebianian, Majid" uniqKey="Tebianian M" first="Majid" last="Tebianian">Majid Tebianian</name><name sortKey="Toghyani, Hadi" sort="Toghyani, Hadi" uniqKey="Toghyani H" first="Hadi" last="Toghyani">Hadi Toghyani</name></noCountry><country name="Iran"><noRegion><name sortKey="Ebrahimi, Seyyed Mahmoud" sort="Ebrahimi, Seyyed Mahmoud" uniqKey="Ebrahimi S" first="Seyyed Mahmoud" last="Ebrahimi">Seyyed Mahmoud Ebrahimi</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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