Expression of Influenza M2e-NP Recombinant Fusion Protein in <i>Escherichia coli</i> BL21 (DE3) and Its Binding to Antibodies
The current influenza vaccines only confer protection against the circulating influenza subtypes, therefore universal vaccines are needed to prevent upcoming influenza outbreaks caused by emerging influenza subtypes. The extracellular domain of influenza A M2 protein (M2e) is highly conserved among...
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MDPI AG
2022-12-01
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| Series: | Vaccines |
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| Online Access: | https://www.mdpi.com/2076-393X/10/12/2066 |
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| author | Mei Peng Tan Noorjahan Banu Mohamed Alitheen Wen Siang Tan Wei Boon Yap |
| author_facet | Mei Peng Tan Noorjahan Banu Mohamed Alitheen Wen Siang Tan Wei Boon Yap |
| author_sort | Mei Peng Tan |
| collection | DOAJ |
| description | The current influenza vaccines only confer protection against the circulating influenza subtypes, therefore universal vaccines are needed to prevent upcoming influenza outbreaks caused by emerging influenza subtypes. The extracellular domain of influenza A M2 protein (M2e) is highly conserved among different subtypes of influenza A viruses, and it is able to elicit protective immunity against the viruses. The influenza nucleoprotein (NP) was used to display the M2e in this study due to its promising T-cell response and adjuvanticity. The <i>M2e</i> gene was fused to the 5′-end of the <i>NP</i> gene and then cloned into pRSET B vector. The DNA sequencing analysis revealed six point mutations in the <i>M2e-NP</i> fusion gene, including one mutation in the M2e peptide and five mutations in the NP. The mutations were reverted using PCR site-directed mutagenesis. The recombinant plasmids (pRSET B-M2e-NP and pRSET B-mM2e-NP) were introduced into <i>Escherichia coli</i> (<i>E. coli</i>) BL21 (DE3) for protein expression. The mutated and non-mutated proteins were subsequently expressed and named mM2e-NP and M2e-NP, respectively. The expression of mM2e-NP and M2e-NP was not affected by the mutations. The binding of anti-M2e antibody to the purified native mM2e-NP and M2e-NP also remained active. However, when the anti-NP antibody was tested, the signal produced by mM2e-NP was very weak. The results implied that the amino acid changes in the NP had adversely impacted on the conformation of mM2e-NP and subsequently affected the antibody binding. In light of the remarkable antibody binding to the M2e-NP fusion protein, this study highly recommends the potential of M2e-NP as a universal influenza vaccine candidate. |
| first_indexed | 2024-03-09T15:46:05Z |
| format | Article |
| id | doaj.art-be9b8aefe1ce4ba5bf26934ad5ab8306 |
| institution | Directory Open Access Journal |
| issn | 2076-393X |
| language | English |
| last_indexed | 2024-03-09T15:46:05Z |
| publishDate | 2022-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Vaccines |
| spelling | doaj.art-be9b8aefe1ce4ba5bf26934ad5ab83062023-11-24T18:32:06ZengMDPI AGVaccines2076-393X2022-12-011012206610.3390/vaccines10122066Expression of Influenza M2e-NP Recombinant Fusion Protein in <i>Escherichia coli</i> BL21 (DE3) and Its Binding to AntibodiesMei Peng Tan0Noorjahan Banu Mohamed Alitheen1Wen Siang Tan2Wei Boon Yap3Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang 43400, MalaysiaDepartment of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang 43400, MalaysiaDepartment of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang 43400, MalaysiaCenter for Toxicology and Health Risk Studies, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, MalaysiaThe current influenza vaccines only confer protection against the circulating influenza subtypes, therefore universal vaccines are needed to prevent upcoming influenza outbreaks caused by emerging influenza subtypes. The extracellular domain of influenza A M2 protein (M2e) is highly conserved among different subtypes of influenza A viruses, and it is able to elicit protective immunity against the viruses. The influenza nucleoprotein (NP) was used to display the M2e in this study due to its promising T-cell response and adjuvanticity. The <i>M2e</i> gene was fused to the 5′-end of the <i>NP</i> gene and then cloned into pRSET B vector. The DNA sequencing analysis revealed six point mutations in the <i>M2e-NP</i> fusion gene, including one mutation in the M2e peptide and five mutations in the NP. The mutations were reverted using PCR site-directed mutagenesis. The recombinant plasmids (pRSET B-M2e-NP and pRSET B-mM2e-NP) were introduced into <i>Escherichia coli</i> (<i>E. coli</i>) BL21 (DE3) for protein expression. The mutated and non-mutated proteins were subsequently expressed and named mM2e-NP and M2e-NP, respectively. The expression of mM2e-NP and M2e-NP was not affected by the mutations. The binding of anti-M2e antibody to the purified native mM2e-NP and M2e-NP also remained active. However, when the anti-NP antibody was tested, the signal produced by mM2e-NP was very weak. The results implied that the amino acid changes in the NP had adversely impacted on the conformation of mM2e-NP and subsequently affected the antibody binding. In light of the remarkable antibody binding to the M2e-NP fusion protein, this study highly recommends the potential of M2e-NP as a universal influenza vaccine candidate.https://www.mdpi.com/2076-393X/10/12/2066influenza Amatrix protein 2 ectodomain (M2e)nucleoprotein (NP)universal vaccineantigenicity |
| spellingShingle | Mei Peng Tan Noorjahan Banu Mohamed Alitheen Wen Siang Tan Wei Boon Yap Expression of Influenza M2e-NP Recombinant Fusion Protein in <i>Escherichia coli</i> BL21 (DE3) and Its Binding to Antibodies Vaccines influenza A matrix protein 2 ectodomain (M2e) nucleoprotein (NP) universal vaccine antigenicity |
| title | Expression of Influenza M2e-NP Recombinant Fusion Protein in <i>Escherichia coli</i> BL21 (DE3) and Its Binding to Antibodies |
| title_full | Expression of Influenza M2e-NP Recombinant Fusion Protein in <i>Escherichia coli</i> BL21 (DE3) and Its Binding to Antibodies |
| title_fullStr | Expression of Influenza M2e-NP Recombinant Fusion Protein in <i>Escherichia coli</i> BL21 (DE3) and Its Binding to Antibodies |
| title_full_unstemmed | Expression of Influenza M2e-NP Recombinant Fusion Protein in <i>Escherichia coli</i> BL21 (DE3) and Its Binding to Antibodies |
| title_short | Expression of Influenza M2e-NP Recombinant Fusion Protein in <i>Escherichia coli</i> BL21 (DE3) and Its Binding to Antibodies |
| title_sort | expression of influenza m2e np recombinant fusion protein in i escherichia coli i bl21 de3 and its binding to antibodies |
| topic | influenza A matrix protein 2 ectodomain (M2e) nucleoprotein (NP) universal vaccine antigenicity |
| url | https://www.mdpi.com/2076-393X/10/12/2066 |
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