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Updated: Aug 16, 2026
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Synthethic peptide used to develop antibodies for detection of polyhedrin from monodon baculovirus (MBV)
Attaphon Satidkanitkul1, Paisarn Sithigorngul, Wiwat Sang-oum
1Department of Biotechnology, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
Abstract:
The use of previously published primers to amplify the monodon baculovirus (MBV) polyhedrin gene sequence by polymerase chain reaction (PCR) from post larvae (PL) of Thai Penaeus monodon resulted in failure. As a result, the putative polyhedrin protein of MBV was isolated from infected PL by homogenization, differential centrifugation and density gradient centrifugation with verification by transmission electron microscopy (TEM). By SDS-PAGE, a single major protein band at 58 kDa was obtained from the putative polyhedrin fraction and this corresponded to a previous report of the molecular weight of polyhedrin from MBV. When used for N-terminal sequence analysis, the putative polyhedrin protein yielded a sequence of 25 amino acids (M F D D S M M M E N M D D L S G D Q K M V L T L A) that did not correspond to the deduced amino acid sequence derived from a previous report of a putative MBV polyhedrin gene amplicon. Despite this, a synthetic peptide of our 25 amino acid sequence (25Pmbv) was conjugated with bovine serum albumin and used as an antigen for antiserum production in mice. Using immunohistochemistry with tissue sections of PL infected with MBV or other viruses, the mouse anti-25Pmbv antiserum showed strong immunoreactivity to occlusion bodies of MBV only. It also showed strong reactivity to the 58 kDa putative polyhedrin protein in Western blots. Altogether, the results suggest that the 58 kDa protein is Thai MBV polyhedrin and that a previously reported MBV polyhedrin gene sequence may represent another protein or polyhedrin from a different variety of MBV.
Insights
Previously published primers failed to amplify the monodon baculovirus (MBV) polyhedrin gene. Researchers isolated a 58 kDa protein, identified as Thai MBV polyhedrin, distinct from prior gene sequence data.
Area of Science:
- Aquaculture virology
- Molecular biology
- Biochemistry
Background:
- Monodon baculovirus (MBV) causes significant losses in shrimp aquaculture.
- Accurate identification of MBV components is crucial for disease management.
- Previous attempts to amplify the MBV polyhedrin gene using published primers were unsuccessful.
Purpose of the Study:
- To isolate and characterize the polyhedrin protein of Thai Monodon baculovirus (MBV).
- To verify the identity of the isolated protein and compare it with existing genetic data.
- To develop a specific antiserum for MBV detection.
Main Methods:
- Isolation of putative polyhedrin protein from infected shrimp post-larvae (PL) via homogenization and centrifugation.
- Protein characterization using SDS-PAGE and N-terminal sequencing.
- Antiserum production using a synthetic peptide and validation via immunohistochemistry and Western blotting.
Main Results:
- A 58 kDa protein was isolated and identified as the putative polyhedrin.
- N-terminal sequencing yielded a 25-amino acid sequence distinct from previously reported gene amplicon data.
- Antiserum raised against the synthetic peptide specifically recognized MBV occlusion bodies and the 58 kDa protein.
Conclusions:
- The 58 kDa protein is identified as the polyhedrin of Thai MBV.
- Discrepancies suggest the previously reported MBV polyhedrin gene sequence may be erroneous or belong to a different MBV strain.
- The developed antiserum is a valuable tool for specific MBV diagnosis in shrimp.
