Synthethic peptide used to develop antibodies for detection of polyhedrin from monodon baculovirus (MBV).
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.
Satidkanitkul
Attaphon
A
Department of Biotechnology, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
Sithigorngul
Paisarn
P
Sang-oum
Wiwat
W
Rukpratanporn
Sombat
S
Sriurairatana
Siriporn
S
Withayachumnankul
Boonsirm
B
Flegel
Timothy
T
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D016367:Baculoviridae / Q000276:immunology*; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera / Q000235:genetics / Q000276:immunology*; D007150:Immunohistochemistry; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000276:immunology; D020539:Sequence Analysis, Protein; D013785:Thailand; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology*
D000907:Antibodies, Bacterial; D007106:Immune Sera; D010455:Peptides; D014764:Viral Proteins