DAO 65:79-84 (2005)  -  doi:10.3354/dao065079

Synthethic peptide used to develop antibodies for detection of polyhedrin from monodon baculovirus (MBV)

Attaphon Satidkanitkul1,2, Paisarn Sithigorngul3, Wiwat Sang-oum2,Sombat Rukpratanporn4, Siriporn Sriurairatana2, Boonsirm Withayachumnankul2,Timothy Flegel1,2,*

1Department of Biotechnology and 2Centex Shrimp, Faculty of Science, Mahidol University, Rama 6 Road,Bangkok 10400, Thailand
3Department of Biology, Faculty of Science, Srinakarinwirot University, Sukhumvit Soi 23, Bangkok 10100, Thailand
4Marine Biotechnology Research Unit and National Center for Genetic Engineering and Biotechnology (BIOTEC),Chulalongkorn University, Phyathai Road, Bangkok 10400, Thailand
*Corresponding author. Email: sctwf@mahidol.ac.th

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.

KEY WORDS: Immunohistochemistry · Monodon baculovirus · MBV · Penaeus monodon · Polyclonal antibody · Polyhedrin

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