tetano
Editor, Senior Moderator
Front Chem. 2018 Feb 13;6:22. doi: 10.3389/fchem.2018.00022. eCollection 2018.
[h=1]Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants.[/h] Firsov A[SUP]1[/SUP], Tarasenko I[SUP]1[/SUP], Mitiouchkina T[SUP]1[/SUP], Shaloiko L[SUP]1[/SUP], Kozlov O[SUP]1[/SUP], Vinokurov L[SUP]1[/SUP], Rasskazova E[SUP]1[/SUP], Murashev A[SUP]1[/SUP], Vainstein A[SUP]2[/SUP], Dolgov S[SUP]1[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3' end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB-M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB-M130 accumulation ranging from 0.25-2.5 μg/g fresh weight (0.0006-0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB-M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB-M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.
[h=4]KEYWORDS:[/h] avian influenza; duckweed; edible vaccine; mice immunization; peptide M2e; ricin B subunit
PMID: 29487846 PMCID: PMC5816751 DOI: 10.3389/fchem.2018.00022
[h=1]Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants.[/h] Firsov A[SUP]1[/SUP], Tarasenko I[SUP]1[/SUP], Mitiouchkina T[SUP]1[/SUP], Shaloiko L[SUP]1[/SUP], Kozlov O[SUP]1[/SUP], Vinokurov L[SUP]1[/SUP], Rasskazova E[SUP]1[/SUP], Murashev A[SUP]1[/SUP], Vainstein A[SUP]2[/SUP], Dolgov S[SUP]1[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3' end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB-M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB-M130 accumulation ranging from 0.25-2.5 μg/g fresh weight (0.0006-0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB-M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB-M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.
[h=4]KEYWORDS:[/h] avian influenza; duckweed; edible vaccine; mice immunization; peptide M2e; ricin B subunit
PMID: 29487846 PMCID: PMC5816751 DOI: 10.3389/fchem.2018.00022