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.

Production and characterization of monoclonal antibodies against mandarinfish ranavirus and first identification of pyloric caecum as the major target tissue

“…Monoclonal antibody has become a very important tool for immunological diagnosis and pathogenesis in the field of aquatic virus due to its high specificity and sensitivity (Aamelfot et al, 2013; Chen et al, 2015, 2016; Sheng et al, 2012). In piscine iridovirus, several mAbs have been developed and characterized (Chen et al, 2016; Nakajima & Sorimachi, 1995; Shi et al, 2003; Vaniksampanna et al, 2023).…”

“…Monoclonal antibody has become a very important tool for immunological diagnosis and pathogenesis in the field of aquatic virus due to its high specificity and sensitivity (Aamelfot et al, 2013; Chen et al, 2015, 2016; Sheng et al, 2012). In piscine iridovirus, several mAbs have been developed and characterized (Chen et al, 2016; Nakajima & Sorimachi, 1995; Shi et al, 2003; Vaniksampanna et al, 2023). Particularly, the mAb M10 against red sea bream iridovirus (RSIV) has been developed as efficient immunofluorescence assay (K Nakajima et al, 1995) and has been widely applied to distinguish RSIV from other piscine iridoviruses worldwide (Gibson‐Kueh et al, 2012; Takano et al, 2020).…”

Imunobiológicos para o diagnóstico laboratorial de ranavirose: clonagem e expressão do gene MCP de isolado brasileiro de >i<Ranavirus>/i< e produção de anticorpos policlonais anti-proteína MCP recombinante

“…Além dos anticorpos policlonais, anticorpos monoclonais têm sido obtidos para a detecção e caracterização de Ranavirus, principalmente na avaliação da expressão de genes durante a replicação viral por ensaios de imunofluorescência (LIN et al, 2014;LIN et al, 2015;CHEN et al, 2016). Lin et al (2014) utilizaram 100 µg da proteína recombinante do MCP (GIV-MCP-His) de Grouper iridovirus (GIV) para imunizar camundongos, sendo coletados os soros policlonais e preparados os hibridomas e, na sequência, selecionados por ELISA (de 73 clones, 6 foram positivos).…”

“…Em anfíbios, diversas infecções por ranaviroses já foram identificadas em animais selvagens e cultivados na Ásia, Américas, Oceania, África e Europa, sendo um dos responsáveis principais pelo declínio mundial dos anuros na natureza (ALENCAR, 2016;CULLEN;OWENS, 2002;CUNNINGHAM et al, 1996;DOCHERTY-BONE et al, 2013;DUFFUS et al, 2017;FOX et al, 2006;GALLI et al, 2006;MAZZONI et al, 2009;OLIVEIRA, 2020;WOLF et al, 1968;ZHANG et al, 2001;ZUPANOVIC et al, 1998). Em salamandras, ocorrências foram registadas na América do Norte e Ásia (BOLLINGER et al, 1999;DOCHERTY et al, 2003;ZHOU et al 2013); e em répteis na Europa, América do Norte, Ásia e Austrália (CHEN;ZHENG;JIANG, 1999;DE VOE et al, 2004;HYATT et al, 2002, HUANG et al, 2009MARSCHANG et al, 1999;WINZELER et al, 2015). Sendo assim, fica evidente sua extensa distribuição global e sua capacidade de causar doenças em várias espécies de vertebrados ectotérmicos (CHINCHAR et al, 2017;DUFFUS et al, 2015).…”