Antibody production with synthetic peptides against human Coronavirus-NL63 ORF3 protein

Human Coronavirus (HCoV) NL63 was detected in an infant in the Netherlands in 2004 and will hereafter be referred to as CoV-NL63. Four years later, it was classified into Group 1 coronaviruses by the International Committee for Taxonomy of Viruses (ICTV), which was modified into alphacoronavirus in 2009. It appears that CoV‐NL63 originates from bats and its hosts are bats and palm civets. A 27,553‐bp CoV‐NL63 genome structure and the spike glycoprotein (S1 domain) use angiotensin‐converting enzyme 2 (ACE2) for infectious entry. Furthermore, CoV‐NL63 simplifies binding to ACE2 by attaching to heparan sulphate proteoglycans on the cell surface. ACE2 has a significant expression in airway differentiated epithelial cells and CoV‐NL63 enter these cells from their apical surface. It is imperative to note that CoV‐NL63, CoV‐229E, CoVHKU1 and CoV‐OC43 infect polarized epithelial cells, and the entry and release of these viruses are related to apical cells in addition to respiratory signs, there are two non-respiratory symptoms including gastroenteritis and Kawasaki disease, both of which are associated with CoV‐NL63. Moreover, neurological manifestations of CoV-NL63 have been reported, especially in children. The main aim of this research study was to generate monoclonal antibodies (mAbs) against the human coronavirus NL63 Open Reading Frame 3 (ORF3) protein. Firstly, bioinformatic tools were used to assist in identifying antigenic determinants and antigenic epitopes were used to generate peptides of ORF3 protein. Thereafter, the ORF3 protein was characterized using various molecular techniques across the bacterial and mammalian expression systems. A CoV-NL63 full-length ORF3 protein (Protein Identity Number: ABE97138.1) was utilized for the synthesis of the peptide from the N-terminus and peptides conjugated with a MAP; KLH and MAP+KLH linkage on the carboxyl end. The mAbs were generated in vivo by immunizing BALB/c mice. Antigen-specific ELISA assessed the antibody specificity and sensitivity. The mAbs that exhibited the highest specificity and sensitivity for antigenic peptide (s) were used to characterize the ORF3 protein by Western blot and immunofluorescence analysis (IFA).

Grantor dc:publisher.institution

University of the Western Cape

Year dc:date.issued

2025

Author dc:creator

• Mnyamana, Yanga Eddie

• Antibody Production
• Synthetic Peptides
• ORF3 Protein
• Coronavirus-NL63
• Human Coronaviruses

Licence

• https://uwcscholar.uwc.ac.za/bitstreams/1f319717-bd21-40f0-a3af-236773c91753/download