The experimental materials used in this study.

<p>The experimental materials used in this study.</p

The stability of M7E_No.2 in F₁ and F₂ of orthogonal (A) and reciprocal (B) cross offspring of Y16 and DS7E (7A).

<p>M:Marker (DL2000); 1: YD-F₁ (A) or DY-F₁ (B); 2–19: YD-F₂ (A) or DY-F₂ (B); 20: Y16; 21: DS7E(7A).</p

The stability of M7E_No.2 in other wheat, amphidiploid and polyploid <i>Th. Elongatum</i>.

<p>M: Marker (DL2000); 1: LD; 2: Y10; 3: Y14; 4: Y16; 5: Y18; 6: Y158; 7: N13; 8: An 8455; 9: Su 3; 10∶8801; 11: <i>Th. elongatum</i> (2<i>n</i> = 4<i>X</i>); 12: <i>Th. elongatum</i> (2<i>n</i> = 10<i>X</i>, PI179162); 13: <i>Th. elongatum</i> (2<i>n</i> = 10<i>X</i>, PI204383); 14: CS; 15: <i>Th. elongatum</i> (2<i>n</i> = 2<i>X</i>).</p

The PCR amplification of M7E_No.1 (A), M7E_No.2 (B) and M7E_No.9 (C) in CS- <i>Thelongatum</i> disomic addition and 7E telodisomic addition lines.

<p>M: Marker (DL2000); 1–7: DA1E-DA7E; 8: CS; 9: <i>Th. elongatum</i> (2<i>n</i> = 2<i>X</i>); 10: DA7ES; 11: DA7EL.</p

The stability of M7E_No.2 in CS- <i>Th. elongatum</i> disomic substitution lines.

<p>M: Marker (DL2000); 1: DS1E (1A); 2: DS1E (1B); 3: DS1E (1D); 4: DS2E (2A); 5: DS2E (2B); 6: DS2E(2D); 7: DS3E(3A); 8: DS3E(3B); 9: DS3E(3D); 10: DS4E(4A); 11: DS4E(4B); 12: DS4E(4D); 13: DS5E(5B); 14: DS5E(5D); 15: DS6E(6A); 16: DS6E(6D); 17: DS7E(7A); 18: DS7E(7B); 19: DS7E(7D); 20: CS; 21: <i>Th. elongatum</i> (2<i>n</i> = 2<i>X</i>).</p

The 7E chromosome-specific molecular markers and PCR primers of <i>Thinopyrum elongatum.</i>

<p>A list of the names of the specific markers is shown, where M7E_No.1 stands for the first (No.1) molecular marker (M) of the <i>Thinopyrum elongatum</i> 7E-chromosome (7E). A list of the name of the specific primers is shown, where P7E_No.1 stands for the first pair (No.1) of primers (P) of the <i>Thinopyrum elongatum</i> 7E-chromosome (7E). Additionally, the name of the original fragments is listed, where SLAF119658 stands for specific (S) length (L) amplified (A) fragment (F), and its number is 119658.</p

The DNA sequences and length of the <i>Thinopyrum elongatum</i> 7E chromosome-specific molecular marker M7E_No.2.

<p>The DNA sequences and length of the <i>Thinopyrum elongatum</i> 7E chromosome-specific molecular marker M7E_No.2.</p

Summary of mouse groups immunized with NM2e.

<p>NS, normal saline.</p>a<p>mice in G1 and G2 were mock-immunized with NS instead of NM2e protein.</p>b<p>mice in G1 and G3 were immunized without adjuvant.</p

Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in <em>E. coli</em>

<div><h3>Background</h3><p>The 23-amino acid extracellular domain of matrix 2 protein (M2e) and the internal nucleoprotein (NP) of influenza are highly conserved among viruses and thus are promising candidate antigens for the development of a universal influenza vaccine. Various M2e- or NP-based DNA or viral vector vaccines have been shown to have high immunogenicity; however, high cost, complicated immunization procedures, and vector-specific antibody responses have restricted their applications. Immunization with an NP–M2e fusion protein expressed in <em>Escherichia coli</em> may represent an alternative strategy for the development of a universal influenza vaccine.</p> <h3>Methodology/Principal Findings</h3><p>cDNA encoding M2e was fused to the 3′ end of NP cDNA from influenza virus A/Beijing/30/95 (H3N2). The fusion protein (NM2e) was expressed in E. coli and isolated with 90% purity. Mice were immunized with recombinant NM2e protein along with aluminum hydroxide gel and/or CpG as adjuvant. NM2e plus aluminum hydroxide gel almost completely protected the mice against a lethal (20 LD₅₀) challenge of heterologous influenza virus A/PR/8/34.</p> <h3>Conclusions/Significance</h3><p>The NM2e fusion protein expressed in <em>E. coli</em> was highly immunogenic in mice. Immunization with NM2e formulated with aluminum hydroxide gel protected mice against a lethal dose of a heterologous influenza virus. Vaccination with recombinant NM2e fusion protein is a promising strategy for the development of a universal influenza vaccine.</p> </div

NM2e protein immunization schedule.

<p>The indicated mice were immunized intramuscularly with NM2e protein with or without adjuvant, three times at 2-week intervals. Blood was collected on days 14, 28, and 38, respectively. The immunized mice were challenged with influenza A virus PR8 at 20-fold the LD₅₀ on day 38. Body weight and survival were monitored for 3 weeks, until day 59.</p