Cell lines
Reagents
Plasmids
Proteins
Pseudotype virus production
Infection assay using pseudotyped viruses
Native EBOV infection inhibition assay under biosafety level 4 conditions
Time-of-addition experiment
Photoaffinity labeling and click chemistry
Photoaffinity labeling and MS measurements
Design and syntheses of eboC24 (C24) peptide
Surface plasmon resonance
Mapping the HR2 domain using amino acid substitutions
Nuclear magnetic resonance
Docking simulation
Production of polyclonal antibodies against the HR2 peptide (KIDQIIHDF)
Detection of the HR2 peptide (KIDQIIHDF)–specific polyclonal antibody
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.