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

Diseases of Aquatic Organisms

Attaphon Satidkanitkul Paisarn Sithigorngul Wiwat Sang-oumSombat Rukpratanporn Siriporn Sriurairatana Boonsirm WithayachumnankulTimothy Flegel¹ 

关键词: Immunohistochemistry;    Monodon baculovirus;    MBV;    Penaeus monodon;    Polyclonal antibody;    Polyhedrin;   

DOI  :  10.3354/dao065079

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

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 putativepolyhedrin 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 wasobtained 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 FD 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 toocclusion 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 genesequence may represent another protein or polyhedrin from a different variety of MBV.

【 授权许可】

Unknown