Gene Cloning and Structural Analyses of the Variable Regions of a Monoclonal Antibody against Foodborne Norovirus
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Abstract:
Foodborne norovirus is an important pathogen that has triggered food safety incidents worldwide. The continued outbreak of COVID-19 has highlighted the urgency of viral safety research in the food sector. In this study, a highly efficient monoclonal antibody against foodborne norovirus obtained by our team was selected and the variable regions of its heavy chain (VH) and light chain (VL) were cloned and analyzed. Total RNA was extracted from the hybridoma cell line 1E3 secreting monoclonal antibodies against foodborne norovirus, and the DNA sequences of the VH and VL genes of the monoclonal antibody 1E3 were amplified by RT-PCR. The fragments were cloned into the PMD19-T vector and sequenced, and the primary amino acid sequences of both variable regions were then analyzed. An NCBI BLAST comparison confirmed that the amplified VH and VL sequences were of mouse antibody variable regions. Using the VBASE2 database to analyze the variable region-encoding gene structures further, the positions of the three amplified complementarity-determining regions and four amplified framework regions on VH and VL were confirmed to be complete. The VH gene was 360 bp long, encoded 120 amino acids, and belonged to the IGHV3-2*02 family. The VL gene was 339 bp long, encoded 113 amino acids, and belonged to the IGKV1-135*01 family. Through molecular docking experiments, it was found that D108 (the key amino acid residue) of the antibody VH chain bonded to N195 of the viral capsid P protein through hydrogen bonding. The successful amplification of the VH and VL fragments of this monoclonal antibody against foodborne norovirus has promoted the development of genetically engineered antibodies and the application of new microbial detection and control technologies for ensuring food safety.
