title: Production and characterization of a monoclonal antibody against a late gene encoded by grouper iridovirus 64L.
abstract: Viral envelope proteins play important roles in viral infection and assembly. The grouper iridovirus ORF 64L (GIV-64L) was predicted to encode an envelope protein and was conserved in all sequenced Ranaviruses. In this study, the complete nucleotide sequence of the GIV-64L gene (1215 bp) was cloned into the isopropyl β-D-1-thiogalactopyranoside (IPTG) induction prokaryotic expression vector pET23a. The approximately 50.2 kDa recombinant GIV-64L-His protein was induced, purified and used as an immunogen to immunize BALB/c mice. Three monoclonal antibodies (mAbs), all IgG1 class antibodies against GIV-64L protein, were produced by enzyme-linked immunosorbent assay. Reverse transcription polymerase chain reaction analyses revealed GIV-64L to be a late gene when expressed in grouper kidney cells during GIV infection with cycloheximide (an inhibitor of protein synthesis) or cytosine arabinoside (an inhibitor of DNA synthesis) present. Finally, one of the established mAbs, GIV-64L-mAb-17, was used in Western blotting and an immunofluorescence assay, which showed that GIV-64L protein was expressed at 24 h post-infection and localized only in the cytoplasm in GIV-infected cells, packed into a whole virus particle. The presently characterized GIV-64L mAbs should have widespread applications in GIV immunodiagnostics and other research, and these results should offer important insights into the pathogenesis of GIV.
authors: Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan.
authors: Z-Y
authors: ZY
authors: Chen
authors: Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan.|Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
authors: P P
authors: PP
authors: Chiou
authors: Department of Nursing, Chang Gung University of Science and Technology, Taoyuan, Taiwan.|Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan.
authors: C-J
authors: CJ
authors: Liou
authors: Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan.
authors: Y-S
authors: YS
authors: Lai
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004266:DNA Virus Infections / Q000175:diagnosis / Q000473:pathology / Q000821:virology*; D005393:Fish Diseases / Q000175:diagnosis / Q000473:pathology / Q000821:virology*; D005399:Fishes; D015967:Gene Expression Regulation, Viral; D017994:Iridovirus / Q000145:classification / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D010802:Phylogeny; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins
keywords: GIV-64L; Grouper iridovirus; immunofluorescence; monoclonal antibody