Abstract
The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.
Highlights
Coronaviruses are a large family of viruses within the family Coronaviridae, and the order Nidovirales, which are mainly divided into four groups
Dulbecco’s minimum essential medium (DMEM) complete medium containing 10% fetal bovine serum and 1% penicillin/streptomycin was used for cell growth and replaced with DMEM without any additives after transfection. 293T cells were used for recombinant receptor-binding domain (RBD) protein expression and SARS-CoV pseudovirus production
SARS-CoV-RBD-specific nanobodies were generated using standard phage display technology from alpacas immunized with recombinant SARS-CoV RBD protein fused with rabbit IgG-Fc tag (RBD-rabbit IgG-Fc protein (rFc)) (Fig. 1A)
Summary
Coronaviruses are a large family of viruses within the family Coronaviridae, and the order Nidovirales, which are mainly divided into four groups. They may cause diseases of different severities in various animals (Zaki et al 2012). There were four sporadic SARS cases reintroduced from animals in Guangdong China in late 2003 and early 2004, of which the virus isolates were different from the previous outbreak (Liang et al 2004). No case has been reported for years, there is a possibility of a new outbreak of SARS. There are no effective antivirals or licensed vaccines to treat or prevent SARS
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