The design of the recombinant peptide eboIZN39IQ (N39) has been previously described (19). The optimized gene encoding eboIZN39IQ (N39) was synthesized by BGI (Beijing, China) and cloned into pET21a (+) vector between the Nde I and Xho I sites to generate pET21-N39. The optimized gene sequence was CACCACCATCATCATCATATTGAAGGCCGCGGCCACATGGATATCAAGAAAGAAATTGAGGCGATCAAGAAAGAGCAGGAAGCGATCAAGAAGAAGATCGAGGCGATCGAGAAAGAACTGCGTCAACTGGCAAACGAAACCACCCAAGCACTGCAACTGTTTCTGCGCGCAACCACCGAACTGCGTACCTTTAGCATCCTGAACCGCAAAGCGATCGATTTTCTGCTGCAGCGCATGAAGCAGATCGAAGACAAGATCGAAGAAATTGAGAGCAAGCAGAAGAAGATCGAGAACGAGATCGCGCGTATCAAAAAACTGATCGGCGAACGTTACTAAA. A 6× His tag was added at the N terminus for affinity purification, followed by the addition of a factor Xa cleavage site (IEGR). BL21 (DE3) was used as the host strain for eboIZN39IQ (N39) expression. The strain hosting pET21-N39 was inoculated into 2 ml of Luria-Bertani (LB) medium containing ampicillin (100 μg/ml) and grown at 37°C while shaking at 220 rpm. The overnight Escherichia coli cultures were then diluted to an optical density (OD) of 0.2 in LB medium and incubated at 37°C. When the culture ODs reached approximately 1.0 (OD600), isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 1 mM. After overnight induction at 30°C, the cells were harvested by centrifugation and resuspended in His-Bind buffer [20 mM tris-HCl (pH 8.0), 250 mM NaCl, and 5 mM imidazole]. The proteins were extracted by passing the cells twice through a microfluidizer at 1200 bar with cooling. The supernatant containing the recombinant peptide eboIZN39IQ (N39) was collected by centrifugation and mixed with Ni-NTA His-Bind resin (Novagen) for 2 hours at 4°C. The unbound proteins were then removed by washing the resin with 10 volumes of wash buffer [20 mM tris-HCl (pH 8.0), 250 mM NaCl, and 20 mM imidazole]. The resin-enriched eboIZN39IQ (N39) peptide was eluted with the same buffer, except that the concentration of imidazole was 500 mM. The purified peptide was dialyzed into 5% acetic acid, further purified by RP-HPLC on a C18 column (GE Healthcare), and then lyophilized. The peptide was then digested with factor Xa protease (New England Biolabs) according to the manufacturer’s protocol. The digested peptide was dialyzed into 5% acetic acid, purified by HPLC, and then lyophilized again. The final peptide sequence was GHMDIKKEIEAIKKEQEAIKKKIEAIEKELRQLANETTQALQLFLRA TTELRTFSILNRKAIDFLLQRMKQIEDKIEEIESKQKKIENEIARIKKLIGERY, where IZm and IQ are shown in bold and the EBOV N-trimer in italics.

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