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Production and characterization of polyclonal antibody against recombinant ORF 049L of rock bream (Oplegnathus fasciatus) iridovirus
Process Biochemistry 42 (2007) 134–140 www.elsevier.com/locate/procbio
Production and characterization of polyclonal antibody against recombinant ORF 049L of rock bream (Oplegnathus fasciatus) iridovirus Yun-Im Kim a, Yu-Mi Ha a, Sang Jung Ahn a, Yoon-Kwon Nam b, Ki-Hong Kim c, Sung-Koo Kim a,* a
Department of Biotechnology and Bioengineering, Pukyong National University, Daeyon 3-dong, Busan 608-737, Republic of Korea b Department of Aquaculture, Pukyong National University, Busan 608-737, Republic of Korea c Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Republic of Korea Received 24 January 2006; received in revised form 4 July 2006; accepted 14 July 2006
Abstract Rock bream iridovirus (RBIV) is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus) in Korea. The structure of the isolated RBIV was observed by an electron microscope, and the virus particles were icosahedral and 120–130 nm in diameter. From the complete genomic DNA sequence of RBIV, the protein encoded in ORF 049L (RBIV-049L) was selected and the property of protein was evaluated with the transmembrane sequence TMHMM 2.0 tool. The ORF 049L gene of RBIV (RBIV-049L) was cloned into pGEX-4T-1 expression vector. The recombinant RBIV-049L was overexpressed in Escherichia coli BL21 (DE3) as a fusion protein (GST-049L, 42 kDa) with a glutathione Stransferase. Antiserum against this recombinant GST-049L protein was prepared in mouse. Dot blot analysis was carried out to identify the reaction abilities and sensitivity of anti-RBIV-049L polyclonal antibody to RBIV-infected rock bream with enzyme linked immunosorbent assay (ELISA) and one-step PCR. These novel RBIV-049L protein and anti-RBIV-049L polyclonal antibody will facilitate the development of more specific and standardized diagnostic techniques. # 2006 Elsevier Ltd. All rights reserved. Keywords: Rock bream iridovirus (RBIV); Rock bream (Oplegnathus fasciatus); ORF-049L; GST-fusion protein; Polyclonal antibody; Transmembrane
1. Introduction Iridoviruses are recognized as causative agents of serious systemic diseases identified from more than 20 fish species in recent years [1–4]. Iridoviruses have icosahedral shape and a large double-stranded DNA genome with 120–300 nm in diameter and contain a spherical deoxyribonucleoprotein core surrounded by a lipid membrane containing transmembrane proteins. The initial signs of the RBIV disease were reduced feed intake, lethargy, and a darkened body with atypical swimming at the edge of cages in the terminal stages of the disease. Characterization of iridoviruses has been hindered because of the difficulty in isolating and propagating the virus in tissue cultures [5–11]. Recently, iridoviral epizootics have occurred frequently among cultured fish in Korea. Iridovirus is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus)
* Corresponding author. Tel.: +82 51 620 6188; fax: +82 51 620 6188. E-mail address: [email protected] (S.-K. Kim). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.07.028
in Korea [11,12], and the complete genome sequence of RBIV has been reported [13]. The genome of RBIV was 112,080 bp long and contained at least 118 putative open reading frames (ORFs). Despite the importance of virus infection in Korea aquaculture industry, researches on vaccines for RBIV have been limited and there are only few studies about antigen analysis of fish iridoviruses [14–16]. Transmembrane (TM) proteins of virus are important functions for cell recognition an epitopes. Therefore, TM proteins of virus are critical targets for drug design, and antigens for the development of vaccines [17]. In the present study, the antigenic properties of the rock bream iridovirus were investigated using polyclonal antibodies against RBIV-049L, the TM protein of RBIV. The transmembrane portion of RBIV-049L was analysed by TMHMM based on a hidden Markov model (HMM) approach [18]. The RBIV specific polyclonal antibody was produced using recombinant ORF 049L protein in mouse. The dot blot analysis, indirect ELISA and one-step PCR combination-detection were carried out to evaluate the reaction sensitivity.
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2. Materials and methods 2.1. Virus samples Tissue specimens of the spleen and kidney obtained from the moribund rock bream infected by RBIV were homogenized in 10 volumes of Eagle’s minimum essential medium. The tissue homogenate was centrifuged at 2500 g for 5 min at 4 8C, and the supernatant of a RBIV sample was passed through a 0.45 mm filter membrane. The permeate was stored at 80 8C as a virus stock until use [19].
2.2. Electron microscopy Isolated viruses were homogenized and centrifuged at 3000 g for 5 min. The pellets were fixed in 2.5% glutaraldehyde for 3 h and post-fixed in 1% OsO4 for 2 h. The fixed sample was dehydrated through a graded ethanol series, infiltrated, and embedded in epoxy resin. Ultrathin sections stained in 2% of uranyl acetate (w/v) and lead citrate (w/v) were examined with a transmission electron microscope (JEOL JEM-1200EX II).
2.3. Extraction of RBIV genomic DNA and amplification For the isolation of RBIV genomic DNA, 20 mg samples of spleen and kidney obtained from rock bream infected by RBIV were prepared, and the genomic DNA of RBIV was extracted with Genomic DNA Extraction Kit (Bioneer Co., Daejeon, Korea). According to the sequence submitted in GenBank database under Accession number AY532606 [13], primers were designed as shown below to amplify the ORF 049L from genomic DNA of rock bream infected by RBIV. EcoRI and SalI restriction sites were incorporated in the forward and reverse primers, respectively, to facilitate cloning in pGEX-4T-1 expression vector (Amersham, Sweden). The amplification product was analyzed by electrophoresis on 1.2% agarose gel stained with ethidium bromide (0.5 mg/ml). Forward primer: 50 -GAATTCATGTACCCTGACTGTCCCAG-30 (EcoRI restriction site). Reverse primer: 50 -GTCGACTTATTTCATAAGCCTTGCACA-30 (SalI restriction site).
2.4. pGEM-T Easy vector subcloning and sequence analysis The PCR product was purified using PCR purification kit (Bioneer Co., Daejeon, Korea) and inserted in pGEM-T Easy vector system (Promega Co., USA). E. coli DH5a was transformed with the ligated plasmid and the transformed cells were cultured using Luria-Bertani (LB) broth containing ampicillin (100 mg/ml). The plasmid DNA was extracted with Plasmid Purification Kit (Nucleogen, Korea). The plasmid DNA was sequenced in Macrogen. Ltd. (Seoul, Korea) and compared with ISKNV (AF371960) and the RBIV (AAT71864) sequences. Nucleotide and predicted amino acid sequences were analyzed using DNAsis (Windows version 2.5, Hitachi software engineering), BioEdit Sequence Alignment Editor (version 5.0.9) and BLAST programs in non-redundant databases of NCBI (http://www.ncbi.nlm.nih.gov/BLAST/).
2.5. Subcloning in expression vector and protein expression RBIV-049L cloned in pGEM-T Easy vector was digested with EcoRI and SalI restriction enzymes and run on 1.2% agarose gel. A 429 bp insert was cut and purified from agarose gel by using Gel Extraction and Purification kit (Qiagen) and cloned in pGEX-4T-1. The recombinant plasmid was named as pGEX-049L and the host E. coli BL21 (DE3) was transformed with the recombinant plasmid. The expression of the fusion gene was induced with 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG). Bacteria cells were collected by centrifugation at 3000 g for 20 min, cells were lysed in 5 sample buffer (60 mM Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, and 0.1% bromophenol blue, with 14.4 mM 2-mercaptoethanol), and analyzed by 12% SDSPAGE.
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To determine the solubility of the recombinant protein, exponential phase culture was induced with 0.4 mM IPTG for 4 h at 25 8C. Cells were harvested by centrifugation at 3000 g for 10 min. The cell pellet was resuspended in 1 ml of buffer A (10 mM Tris–HCl, pH 7.5). The cell suspension was sonicated on ice. The obtained cell lysate was fractionated as soluble and insoluble fraction after centrifuged at 16,000 g for 30 min. The insoluble fraction was resuspended in 1 ml of buffer A. Soluble and insoluble fractions were then analyzed on 12% SDS-PAGE.
2.6. Production of polyclonal antibodies against the recombinant protein The soluble protein was purified by affinity chromatography using a GSTrapFF (Amersham, Sweden) column according to the manufacturer’s instructions. Two groups of BALB/c mice were prepared for non-immunization group and recombinant GST-049L vaccine group. The antigen (100 mg aliquots) was mixed with equal volume of Freund’s complete adjuvant (Sigma Chemical Co., USA). The emulsion was injected intradermally into 6-week-old female BALB/c mice (Hyochang Science, Daegu, Korea). Booster containing 100 mg of antigen mixed with Freund’s incomplete adjuvant (Sigma Chemical Co., USA) was injected three times at a week intervals. Sarcoma cells (1 107 cells/ml) were injected intraperitoneally into the mice 1 week after the final booster. Ascetic fluid was collected after 10–15 day, clarified by overnight incubation at 4 8C, and centrifuged at 3000 g for 15 min. The supernatant was collected stored at 20 8C.
2.7. Western blotting Expressed recombinant proteins were separated on 12% SDS-PAGE. After electrophoresis at 90 V, one loaded gel was stained with Brilliant Blue G (Sigma, USA). The other gel was transferred onto a 0.45 mm pore nitrocellulose membrane (BioTrace, PALL, USA) at 100 V for 1 h in a Bio-Rad mini TransBlot electrophoretic transfer cell for western blot analysis. The blotted membrane was rinsed with TTBS (0.02 M Tris–HCl, 0.5 M NaCl, 0.05 % Tween-20, pH 7.5) three times for 10 min and then blocked in TBS (0.02 M Tris–HCl, 0.5 M NaCl, pH 7.5) containing 3% (w/v) BSA for 2 h at room temperature and overnight at 4 8C. Then, the membrane was rinsed with TTBS three times for 10 min and this membrane was put into mice polyclonal antisera diluted 1:2000 in TTBS containing 1% BSA, and incubated for 2 h at room temperature. After rinsing of the membrane three times for each 15 min in TTBS, the membrane was treated for 1 h with alkaline phosphatase conjugated anti-mouse IgG (1:2000, Santa Cruz Biotechnology, USA) in TTBS containing 1% BSA. Then the membrane was washed three times for 10 min in TTBS and developed by BCIP/NBT (Sigma, USA) for 1–3 min. The development reaction was stop by rinsing strips with distilled water.
2.8. Dot blot analysis of RBIV-049L in membrane and cytosol extracts from the virus-infected rock bream For the detection of RBIV-049L antigen from virus-infected fish, tissue specimens of the spleen and kidney obtained from the moribund rock bream infected by RBIV were homogenized in 10 volumes of homogenizing buffer (20 mM Tris (pH 7.5), 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). The lysate was centrifuged at 1000 g for 10 min at 4 8C to remove cell wall debris. The supernatant was collected and ultracentrifuged at 100,000 g for 1 h at 4 8C. The supernatant (cytosol) was collected and the resulting pellet (crude membranes) was resuspended in homogenizing buffer with 1% Triton X-100 for 1 h at 4 8C. Three fractions of membrane extracts from virus-infected rock bream were obtained after centrifugation at 100,000 g for 1 h at 4 8C. Top layer with white fraction (IM 1), middle layer with transparent solution (IM 2) and bottom layer with turbidity (IM 3) were obtained. For the detection of RBIV in membrane fraction 1, 2, 3 (except in normal rock bream) and cytosol extracts from virus-infected fish, isolated total DNA from each extracts were used as templates for one-step PCR amplification. The procedure used for the dot blot analysis was followed by Lu et al. and Nadala and Loh [20,21]. The nitrocellulose (NC) membrane (Bio-Rad, USA) was cut to the desired size and soaked in PBS for 15 min. The membrane was mounted onto a dot blot apparatus (model DP-96; Advantec, JAPAN). The
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concentrations of antigens from total virus, membrane and cytosol fractions from virus-infected rock bream were determined with Bradford assay. A serial dilution with phosphate-buffered saline from 300 mg to 3 ng of virus protein samples were spotted onto NC membrane and dried. Membrane and cytosol proteins from uninfected rock bream and recombinant GST-049L protein were also used as negative and positive controls. The NC membrane was incubated in blocking solution-I (PBS containing 1% gelatin) for 2 h at room temperature. The membrane was treated with the mouse anti-RBIV-049L (1:200 in blocking solution-I) for 2 h at room temperature. The membrane was washed with PBS-T (PBS with 0.05% Tween-20), followed by incubation with alkaline phosphatase conjugated anti-mouse IgG (1:2000, Santa Cruz Biotechnology, USA) for 1 h at room temperature. Then, the membrane was washed three times for 10 min in PBS-T and developed by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma, USA) for 1–2 min. The development reaction was stopped rinsing strips with distilled water.
2.9. Enzyme linked immunosorbent assay (ELISA) Well plate (96 well) was coated with 50 ml of RBIV stock and incubated at room temperature for overnight. The plate was then rinsed three times with PBS-T and incubated with blocking solution-II (PBS containing 3% (w/v) skimmed milk) at 37 8C for 2 h. After washing with PBS-T, 100 ml of a 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 or 1:3200 dilution of blocking solution-II containing anti-RBIV-049L. After 3-h incubation at room temperature, plates were rinsed three times with PBS-T, and then 100 ml of 1:2000-diluted Horse Radish Peroxidase (HRP)-anti-mouse (Sigma, USA) was added to each well and incubated for 1 h at room temperature. After washing 8 times, the enzyme activity was determined by adding 100 ml of 3,30 ,5,50 -Tetramethylbenzidine substrate solution. Optical density was read at 630 nm with an ELISA plate reader after incubating for 30 min. The procedure used for the indirect ELISA was followed by Cliquet et al. [22]. Well plate was coated with 300 mg to 3 ng of protein samples and incubated at room temperature overnight. Membrane and cytosol proteins from uninfected rock bream and recombinant GST-049L protein were also used as negative and positive controls. The plate was then rinsed three times with PBS-T and incubated with blocking solution-II at 37 8C for 2 h. After washing, 100 ml of 1:200 dilution of blocking solution-II containing anti-RBIV-049L were added to each well. ELISA detection was performed as described above.
3. Results and discussion 3.1. Electron microscopy The structure of the isolated RBIV was observed with an electron microscope (Fig. 1). Electron microscopy of ultrathin sections revealed large number virus particles in the spleen tissue. The virus particles were icosahedral and 120–130 nm in diameter, which are typical features of Iridoviridae viruses [23,24]. The host range and morphological characteristics indicated that the isolated viruses belong to the Iridoviridae family.
Fig. 1. Isolated RBIVobserved with an electron microscope. The virus particles in infected rock bream were icosahedral and 120–130 nm in diameter, which are typical features of iridoviridae viruses.
contained putative transmembrane helices was analyzed with ExPASy Proteomics tools, Prediction of transmembrane helices in proteins (TMHMM Server v. 2.0: http://kr.expasy.org/tools/) as shown in Fig. 3. Comparing the RBIV-049L to the sequences in the GenBank databases, RBIV-049L had high degree of identity with other Megalocytivirus species, Orange-spotted grouper iridovirus ORF52L (AAX82361) and infectious spleen and kidney necrosis virus (ISKNV) ORF050L (AAL98774) (93–99%) as shown in Fig. 4. Interestingly, low degree of identity with other Iridoviridae family except Orange-spotted grouper iridovirus and infectious spleen and kidney necrosis virus was observed in RBIV-049L by BLASTn and BLASTx searches of the GenBank databases.
3.2. Cloning and characterization of RBIV-049L The PCR product, using primers for the RBIV-049L gene, was produced as a DNA fragment of 429 bp, as shown in Fig. 2. The RBIV-049L gene was cloned in the pGEM-T Easy vector system by PCR and confirmed by DNA sequencing (Macrogen. Ltd., Seoul, Korea). The cloned sequence was entirely identical to the published sequence of RBIV-049L in the GenBank database (Accession number AY532606). The nucleotide sequence of RBIV-049L was predicted to encode an open reading frame of 142 amino acids, which
3.3. Expression and purification of recombinant protein GST-049L Small-scale cultures of the positive clones (selected on the basis of PCR screening) were subjected to IPTG induction to identify clones capable of expressing the predicted 42 kDa recombinant protein. The RBIV-049L gene was expressed in E. coli as a fusion protein with 26 kDa of glutathione Stransferase. From the analysis of SDS-PAGE, correct size recombinant protein was observed as a 42 kDa fusion protein
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Fig. 2. Detection of the target gene isolated from RBIV by PCR (A) and construction of recombinant plasmid pGEX-049L (B). (A) M1: 100 bp DNA ladder, lane 1: ORF 049L gene from RBIV; (B) M1: 100 bp DNA ladder, lane 1: pGEX-049L digested with EcoRI, lane 2: pGEX-049L digested with SalI, lane 3: pGEX-049L digested with EcoRI and SalI, M2: 1 kb DNA ladder.
(Fig. 5: panel A, lane 5). The results confirmed that the ORF049L was selectively expressed in the transformed E. coli BL21 (DE3) cells. Protein expression was not observed in the negative control, E. coli BL21 (DE3) cells, E. coli BL21 (DE3) cells transformed with the pGEX-4T-1 (induction), and E. coli BL21 (DE3) cells transformed with the pGEX-049L (without induction). The expression level of GST-049L fusion protein increased with the induction time. The highest amount of fusion protein was produced at 4 h after 0.4 mM IPTG induction. The soluble GST-049L fusion protein was applied to GSTrapFF affinity column chromatography, the band of GST-049L fusion protein showed high purities and correct size by SDS-PAGE (Fig. 5: panel A, lane 8).
adjuvant. Western blot was carried out to identify the reaction abilities of anti-RBIV-049L polyclonal antibody to recombinant RBIV-049L. Recombinant RBIV-049L protein could be detect by anti-RBIV-049L polyclonal antibody. (Fig. 5: panel B). Also, ELISA was performed to determine the proper dilution of antiserum against GST-049L. Anti-RBIV-049L polyclonal antibody could be detectable with 800 times dilution of the original polyclonal antibody solution (Fig. 6). However, 200 dilution was used for the detection of RBIV-049L for safety from the error.
3.4. Production of RBIV-049L-specific polyclonal antibody
RBIV in membrane fraction 1 from RBIV-infected rock breams (Fig. 7(A) lane 7, IM 1) could be detected by dot blot analysis using antiserum to GST-049L. The strongest reaction was observed in positive lane 1 of GST-049L. Strong signals were also observed in lane 5, 7, and 8 of total protein and membrane fraction 1 and 2 from RBIV-infected rock breams, respectively. Also, one-step PCR was performed to compare the sensitivity of dot blot analysis (Fig. 7(B)). Thick bands were observed in lane 1, 3, and 4 of DNA samples extracted from virus total protein and membrane fraction 1 and 2. To investigate the specificity and sensitivity of anti-RBIV049L serum against RBIV, the indirect ELISA assay was performed in total, cytosol and membrane fractions from RBIVinfected rock breams. As shown in Fig. 8, the indirect ELISA using anti-RBIV-049L serum recognized the total protein and membrane fraction 1 from RBIV-infected rock breams. Recent reports have suggested that direct PCR and one-step PCR (including two-step PCR) be used as sensitive and specific alternative protocols to western blot assay for the detection of fish viruses without sacrificing the animals [25,26]. Immunoblot detection system and one-step PCR combination-detection system would be useful to investigate the specificities and sensitivities of antibodies against fish viruses
BALB/c mice were immunized by an intraperitoneal injection with 100 mg of purified recombinant GST-049L emulsified with an equal volume of Freund’s complete
Fig. 3. Predicted membrane topology of RBIV-049L protein. Transmembrane helix predictions were performed with the computational method using the TMHMM Server v. 2.0. The graphs show the probability of a given amino acid residue of RBIV-049L as a transmembrane helix. The expected number of amino acids in transmembrane helices is 21.5. If this number is larger than 18 it is very likely to be a transmembrane protein (http://www.cbs.dtu.dk/services/ TMHMM-2.0/).
3.5. Detection of RBIV-049L from RBIV-infected rock breams
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Fig. 4. Comparisons of the nucleotides and amino acids sequences of RBIV-ORF049L to those of RBIV-049L-like ORFs of other Megalocytivirus species. (A) Alignment of the nucleotide sequences of PCR product from RBIV and other Megalocytivirus species. (B) Comparison of the amino acid sequence of RBIVORF049L to that of Orange-spotted grouper iridovirus ORF052L and infectious spleen and kidney necrosis virus ORF050L. Identical amino acid residues are darkly shaded, similar amino acids are lightly shaded. The alignment was created using the BioEdit Sequence Alignment Editor version 5.0.9 using the Clustal W algorithm. GenBank Accession numbers: Rock bream iridovirus (Rock bream iridovirus strain RBIV-KOR-TY1 from South Korea), AY532606; Orange-spotted grouper iridovirus, AY894343; infectious spleen and kidney necrosis virus, AF371960.
Fig. 5. Expression of fusion GST-049L protein in E. coli BL21 (DE3) and western blotting. (A) M: low molecular marker, lane 1: total protein of E. coli BL21 (DE3), lane 2: total protein of non-induced pGEX-4T-1, lane 3: total protein of induced pGEX-4T-1, lane 4: total protein of non-induced pGEX-049L, lane 5: total protein of induced pGEX-049L, lane 6: soluble protein of induced pGEX-049L, lane 7: insoluble protein of induced pGEX-049L, lane 8: purified protein of pGEX-049L (B) M: mid-range prestained protein size marker, lane 1: expressed GST-049L protein reacted with non-immunized mice serum (negative control), lane 2: non-induced GST049L protein reacted with anti-RBIV-049L polyclonal antibody (negative control), lane 3: expressed protein of GST-049L protein reacted with anti-RBIV-049L polyclonal antibody.
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Fig. 6. Determination of anti-RBIV-049L serum dilution concentration for indirect ELISA for RBIV detection.
Fig. 8. Detection of RBIV by indirect ELISA from virus-infected rock breams.
[27,28]. Considering the results, it could be suggested that dot blot detection system, indirect ELISA and one-step PCR combination-detection system be useful method for sensitive and specific detection of RBIV. Subcellular localization of virus in RBIV-infected rock breams evaluated by dot blot analysis,
indirect ELISA and one-step PCR detection system (Figs. 7 and 8). Direct amplification (direct PCR) from serum or blood samples using ORF 049L primers showed lower sensitivity than those of one-step PCR. Membrane fraction 1 (Fig. 7(A) lane 7, IM 1) from RBIVinfected rock breams showed strongest signals in the combination-detection system and the ORF 049L was located within the membrane fraction 1 from RBIV-infected rock breams. 4. Conclusions
Fig. 7. Sensitivity of the anti-RBIV-049L in dot blot analysis compared with one-step PCR 049L from RBIV-infected rock breams. (A) lane 1 (GST-049L): positive control, purified protein of pGEX-049L, lane 2 (NT): total protein of normal (uninfected) rock bream, lane 3 (NC): cytosol of normal rock bream, lane 4 (NM): membrane extracts of normal rock bream, lane 5 (IT): total protein of RBIV-infected rock bream, lane 6 (IC): cytosol of RBIV-infected rock bream, lane 7 (IM 1): membrane extract 1 (virus band) of RBIV-infected rock bream, lane 8 (IM 2): membrane extract of RBIV-infected rock bream, lane 9 (IM 3): membrane extract of RBIV-infected rock bream: lane 10 (BSA): negative control, lane A–F: total protein range from 300 to 0.003 mg (GST-049L: 3 mg to 0.03 ng). (B) One-step PCR of total genomic DNA from total (IT), cytosol extract (IC) and membrane extracts (IM 1, IM 2, IM 3) from the virusinfected rock bream.
The RBIV-specific polyclonal antibody was produced and characterized in this study. This paper is the first to report the overexpression and purification of the novel 16 kDa molecular protein, ORF 049L of RBIV, which is expected as a transmembrane protein of RBIV. The pGEX-4T-1 expression system was used for overexpression of RBIV-049L. The RBIV specific polyclonal antibody was produced using recombinant ORF 049L protein in mouse. Western blot was carried out to evaluate the reaction abilities of anti-RBIV-049L polyclonal antibody to GST-049L. Sensitivity and specificity of the antiRBIV-049L polyclonal antibody for the detection of RBIV antigens were improved by combination-detection system using dot blot analysis, indirect ELISA, and one-step PCR. ORF 049L located within the membrane fraction 1 from RBIVinfected rock breams. Furthermore, one-step PCR with dot blotindirect ELISA combination-detection system using ORF 049L and its antiserum showed sensitivity and specificity for the detection of RBIV. Our present results indicate that it can be possible to develop an expressed gene-based specific therapy against RBIV. Further studies are carried out for the evaluation of structure and function of the RBIV-049L protein. Acknowledgments This research was supported by Regional Research Fund from the Ministry of Commerce, Industry and Energy. Yun-Im
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Production and characterization of polyclonal antibody against recombinant ORF 049L of rock bream (Oplegnathus fasciatus) iridovirus Yun-Im Kim a, Yu-Mi Ha a, Sang Jung Ahn a, Yoon-Kwon Nam b, Ki-Hong Kim c, Sung-Koo Kim a,* a
Department of Biotechnology and Bioengineering, Pukyong National University, Daeyon 3-dong, Busan 608-737, Republic of Korea b Department of Aquaculture, Pukyong National University, Busan 608-737, Republic of Korea c Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Republic of Korea Received 24 January 2006; received in revised form 4 July 2006; accepted 14 July 2006
Abstract Rock bream iridovirus (RBIV) is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus) in Korea. The structure of the isolated RBIV was observed by an electron microscope, and the virus particles were icosahedral and 120–130 nm in diameter. From the complete genomic DNA sequence of RBIV, the protein encoded in ORF 049L (RBIV-049L) was selected and the property of protein was evaluated with the transmembrane sequence TMHMM 2.0 tool. The ORF 049L gene of RBIV (RBIV-049L) was cloned into pGEX-4T-1 expression vector. The recombinant RBIV-049L was overexpressed in Escherichia coli BL21 (DE3) as a fusion protein (GST-049L, 42 kDa) with a glutathione Stransferase. Antiserum against this recombinant GST-049L protein was prepared in mouse. Dot blot analysis was carried out to identify the reaction abilities and sensitivity of anti-RBIV-049L polyclonal antibody to RBIV-infected rock bream with enzyme linked immunosorbent assay (ELISA) and one-step PCR. These novel RBIV-049L protein and anti-RBIV-049L polyclonal antibody will facilitate the development of more specific and standardized diagnostic techniques. # 2006 Elsevier Ltd. All rights reserved. Keywords: Rock bream iridovirus (RBIV); Rock bream (Oplegnathus fasciatus); ORF-049L; GST-fusion protein; Polyclonal antibody; Transmembrane
1. Introduction Iridoviruses are recognized as causative agents of serious systemic diseases identified from more than 20 fish species in recent years [1–4]. Iridoviruses have icosahedral shape and a large double-stranded DNA genome with 120–300 nm in diameter and contain a spherical deoxyribonucleoprotein core surrounded by a lipid membrane containing transmembrane proteins. The initial signs of the RBIV disease were reduced feed intake, lethargy, and a darkened body with atypical swimming at the edge of cages in the terminal stages of the disease. Characterization of iridoviruses has been hindered because of the difficulty in isolating and propagating the virus in tissue cultures [5–11]. Recently, iridoviral epizootics have occurred frequently among cultured fish in Korea. Iridovirus is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus)
* Corresponding author. Tel.: +82 51 620 6188; fax: +82 51 620 6188. E-mail address: [email protected] (S.-K. Kim). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.07.028
in Korea [11,12], and the complete genome sequence of RBIV has been reported [13]. The genome of RBIV was 112,080 bp long and contained at least 118 putative open reading frames (ORFs). Despite the importance of virus infection in Korea aquaculture industry, researches on vaccines for RBIV have been limited and there are only few studies about antigen analysis of fish iridoviruses [14–16]. Transmembrane (TM) proteins of virus are important functions for cell recognition an epitopes. Therefore, TM proteins of virus are critical targets for drug design, and antigens for the development of vaccines [17]. In the present study, the antigenic properties of the rock bream iridovirus were investigated using polyclonal antibodies against RBIV-049L, the TM protein of RBIV. The transmembrane portion of RBIV-049L was analysed by TMHMM based on a hidden Markov model (HMM) approach [18]. The RBIV specific polyclonal antibody was produced using recombinant ORF 049L protein in mouse. The dot blot analysis, indirect ELISA and one-step PCR combination-detection were carried out to evaluate the reaction sensitivity.
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2. Materials and methods 2.1. Virus samples Tissue specimens of the spleen and kidney obtained from the moribund rock bream infected by RBIV were homogenized in 10 volumes of Eagle’s minimum essential medium. The tissue homogenate was centrifuged at 2500 g for 5 min at 4 8C, and the supernatant of a RBIV sample was passed through a 0.45 mm filter membrane. The permeate was stored at 80 8C as a virus stock until use [19].
2.2. Electron microscopy Isolated viruses were homogenized and centrifuged at 3000 g for 5 min. The pellets were fixed in 2.5% glutaraldehyde for 3 h and post-fixed in 1% OsO4 for 2 h. The fixed sample was dehydrated through a graded ethanol series, infiltrated, and embedded in epoxy resin. Ultrathin sections stained in 2% of uranyl acetate (w/v) and lead citrate (w/v) were examined with a transmission electron microscope (JEOL JEM-1200EX II).
2.3. Extraction of RBIV genomic DNA and amplification For the isolation of RBIV genomic DNA, 20 mg samples of spleen and kidney obtained from rock bream infected by RBIV were prepared, and the genomic DNA of RBIV was extracted with Genomic DNA Extraction Kit (Bioneer Co., Daejeon, Korea). According to the sequence submitted in GenBank database under Accession number AY532606 [13], primers were designed as shown below to amplify the ORF 049L from genomic DNA of rock bream infected by RBIV. EcoRI and SalI restriction sites were incorporated in the forward and reverse primers, respectively, to facilitate cloning in pGEX-4T-1 expression vector (Amersham, Sweden). The amplification product was analyzed by electrophoresis on 1.2% agarose gel stained with ethidium bromide (0.5 mg/ml). Forward primer: 50 -GAATTCATGTACCCTGACTGTCCCAG-30 (EcoRI restriction site). Reverse primer: 50 -GTCGACTTATTTCATAAGCCTTGCACA-30 (SalI restriction site).
2.4. pGEM-T Easy vector subcloning and sequence analysis The PCR product was purified using PCR purification kit (Bioneer Co., Daejeon, Korea) and inserted in pGEM-T Easy vector system (Promega Co., USA). E. coli DH5a was transformed with the ligated plasmid and the transformed cells were cultured using Luria-Bertani (LB) broth containing ampicillin (100 mg/ml). The plasmid DNA was extracted with Plasmid Purification Kit (Nucleogen, Korea). The plasmid DNA was sequenced in Macrogen. Ltd. (Seoul, Korea) and compared with ISKNV (AF371960) and the RBIV (AAT71864) sequences. Nucleotide and predicted amino acid sequences were analyzed using DNAsis (Windows version 2.5, Hitachi software engineering), BioEdit Sequence Alignment Editor (version 5.0.9) and BLAST programs in non-redundant databases of NCBI (http://www.ncbi.nlm.nih.gov/BLAST/).
2.5. Subcloning in expression vector and protein expression RBIV-049L cloned in pGEM-T Easy vector was digested with EcoRI and SalI restriction enzymes and run on 1.2% agarose gel. A 429 bp insert was cut and purified from agarose gel by using Gel Extraction and Purification kit (Qiagen) and cloned in pGEX-4T-1. The recombinant plasmid was named as pGEX-049L and the host E. coli BL21 (DE3) was transformed with the recombinant plasmid. The expression of the fusion gene was induced with 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG). Bacteria cells were collected by centrifugation at 3000 g for 20 min, cells were lysed in 5 sample buffer (60 mM Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, and 0.1% bromophenol blue, with 14.4 mM 2-mercaptoethanol), and analyzed by 12% SDSPAGE.
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To determine the solubility of the recombinant protein, exponential phase culture was induced with 0.4 mM IPTG for 4 h at 25 8C. Cells were harvested by centrifugation at 3000 g for 10 min. The cell pellet was resuspended in 1 ml of buffer A (10 mM Tris–HCl, pH 7.5). The cell suspension was sonicated on ice. The obtained cell lysate was fractionated as soluble and insoluble fraction after centrifuged at 16,000 g for 30 min. The insoluble fraction was resuspended in 1 ml of buffer A. Soluble and insoluble fractions were then analyzed on 12% SDS-PAGE.
2.6. Production of polyclonal antibodies against the recombinant protein The soluble protein was purified by affinity chromatography using a GSTrapFF (Amersham, Sweden) column according to the manufacturer’s instructions. Two groups of BALB/c mice were prepared for non-immunization group and recombinant GST-049L vaccine group. The antigen (100 mg aliquots) was mixed with equal volume of Freund’s complete adjuvant (Sigma Chemical Co., USA). The emulsion was injected intradermally into 6-week-old female BALB/c mice (Hyochang Science, Daegu, Korea). Booster containing 100 mg of antigen mixed with Freund’s incomplete adjuvant (Sigma Chemical Co., USA) was injected three times at a week intervals. Sarcoma cells (1 107 cells/ml) were injected intraperitoneally into the mice 1 week after the final booster. Ascetic fluid was collected after 10–15 day, clarified by overnight incubation at 4 8C, and centrifuged at 3000 g for 15 min. The supernatant was collected stored at 20 8C.
2.7. Western blotting Expressed recombinant proteins were separated on 12% SDS-PAGE. After electrophoresis at 90 V, one loaded gel was stained with Brilliant Blue G (Sigma, USA). The other gel was transferred onto a 0.45 mm pore nitrocellulose membrane (BioTrace, PALL, USA) at 100 V for 1 h in a Bio-Rad mini TransBlot electrophoretic transfer cell for western blot analysis. The blotted membrane was rinsed with TTBS (0.02 M Tris–HCl, 0.5 M NaCl, 0.05 % Tween-20, pH 7.5) three times for 10 min and then blocked in TBS (0.02 M Tris–HCl, 0.5 M NaCl, pH 7.5) containing 3% (w/v) BSA for 2 h at room temperature and overnight at 4 8C. Then, the membrane was rinsed with TTBS three times for 10 min and this membrane was put into mice polyclonal antisera diluted 1:2000 in TTBS containing 1% BSA, and incubated for 2 h at room temperature. After rinsing of the membrane three times for each 15 min in TTBS, the membrane was treated for 1 h with alkaline phosphatase conjugated anti-mouse IgG (1:2000, Santa Cruz Biotechnology, USA) in TTBS containing 1% BSA. Then the membrane was washed three times for 10 min in TTBS and developed by BCIP/NBT (Sigma, USA) for 1–3 min. The development reaction was stop by rinsing strips with distilled water.
2.8. Dot blot analysis of RBIV-049L in membrane and cytosol extracts from the virus-infected rock bream For the detection of RBIV-049L antigen from virus-infected fish, tissue specimens of the spleen and kidney obtained from the moribund rock bream infected by RBIV were homogenized in 10 volumes of homogenizing buffer (20 mM Tris (pH 7.5), 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). The lysate was centrifuged at 1000 g for 10 min at 4 8C to remove cell wall debris. The supernatant was collected and ultracentrifuged at 100,000 g for 1 h at 4 8C. The supernatant (cytosol) was collected and the resulting pellet (crude membranes) was resuspended in homogenizing buffer with 1% Triton X-100 for 1 h at 4 8C. Three fractions of membrane extracts from virus-infected rock bream were obtained after centrifugation at 100,000 g for 1 h at 4 8C. Top layer with white fraction (IM 1), middle layer with transparent solution (IM 2) and bottom layer with turbidity (IM 3) were obtained. For the detection of RBIV in membrane fraction 1, 2, 3 (except in normal rock bream) and cytosol extracts from virus-infected fish, isolated total DNA from each extracts were used as templates for one-step PCR amplification. The procedure used for the dot blot analysis was followed by Lu et al. and Nadala and Loh [20,21]. The nitrocellulose (NC) membrane (Bio-Rad, USA) was cut to the desired size and soaked in PBS for 15 min. The membrane was mounted onto a dot blot apparatus (model DP-96; Advantec, JAPAN). The
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concentrations of antigens from total virus, membrane and cytosol fractions from virus-infected rock bream were determined with Bradford assay. A serial dilution with phosphate-buffered saline from 300 mg to 3 ng of virus protein samples were spotted onto NC membrane and dried. Membrane and cytosol proteins from uninfected rock bream and recombinant GST-049L protein were also used as negative and positive controls. The NC membrane was incubated in blocking solution-I (PBS containing 1% gelatin) for 2 h at room temperature. The membrane was treated with the mouse anti-RBIV-049L (1:200 in blocking solution-I) for 2 h at room temperature. The membrane was washed with PBS-T (PBS with 0.05% Tween-20), followed by incubation with alkaline phosphatase conjugated anti-mouse IgG (1:2000, Santa Cruz Biotechnology, USA) for 1 h at room temperature. Then, the membrane was washed three times for 10 min in PBS-T and developed by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma, USA) for 1–2 min. The development reaction was stopped rinsing strips with distilled water.
2.9. Enzyme linked immunosorbent assay (ELISA) Well plate (96 well) was coated with 50 ml of RBIV stock and incubated at room temperature for overnight. The plate was then rinsed three times with PBS-T and incubated with blocking solution-II (PBS containing 3% (w/v) skimmed milk) at 37 8C for 2 h. After washing with PBS-T, 100 ml of a 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 or 1:3200 dilution of blocking solution-II containing anti-RBIV-049L. After 3-h incubation at room temperature, plates were rinsed three times with PBS-T, and then 100 ml of 1:2000-diluted Horse Radish Peroxidase (HRP)-anti-mouse (Sigma, USA) was added to each well and incubated for 1 h at room temperature. After washing 8 times, the enzyme activity was determined by adding 100 ml of 3,30 ,5,50 -Tetramethylbenzidine substrate solution. Optical density was read at 630 nm with an ELISA plate reader after incubating for 30 min. The procedure used for the indirect ELISA was followed by Cliquet et al. [22]. Well plate was coated with 300 mg to 3 ng of protein samples and incubated at room temperature overnight. Membrane and cytosol proteins from uninfected rock bream and recombinant GST-049L protein were also used as negative and positive controls. The plate was then rinsed three times with PBS-T and incubated with blocking solution-II at 37 8C for 2 h. After washing, 100 ml of 1:200 dilution of blocking solution-II containing anti-RBIV-049L were added to each well. ELISA detection was performed as described above.
3. Results and discussion 3.1. Electron microscopy The structure of the isolated RBIV was observed with an electron microscope (Fig. 1). Electron microscopy of ultrathin sections revealed large number virus particles in the spleen tissue. The virus particles were icosahedral and 120–130 nm in diameter, which are typical features of Iridoviridae viruses [23,24]. The host range and morphological characteristics indicated that the isolated viruses belong to the Iridoviridae family.
Fig. 1. Isolated RBIVobserved with an electron microscope. The virus particles in infected rock bream were icosahedral and 120–130 nm in diameter, which are typical features of iridoviridae viruses.
contained putative transmembrane helices was analyzed with ExPASy Proteomics tools, Prediction of transmembrane helices in proteins (TMHMM Server v. 2.0: http://kr.expasy.org/tools/) as shown in Fig. 3. Comparing the RBIV-049L to the sequences in the GenBank databases, RBIV-049L had high degree of identity with other Megalocytivirus species, Orange-spotted grouper iridovirus ORF52L (AAX82361) and infectious spleen and kidney necrosis virus (ISKNV) ORF050L (AAL98774) (93–99%) as shown in Fig. 4. Interestingly, low degree of identity with other Iridoviridae family except Orange-spotted grouper iridovirus and infectious spleen and kidney necrosis virus was observed in RBIV-049L by BLASTn and BLASTx searches of the GenBank databases.
3.2. Cloning and characterization of RBIV-049L The PCR product, using primers for the RBIV-049L gene, was produced as a DNA fragment of 429 bp, as shown in Fig. 2. The RBIV-049L gene was cloned in the pGEM-T Easy vector system by PCR and confirmed by DNA sequencing (Macrogen. Ltd., Seoul, Korea). The cloned sequence was entirely identical to the published sequence of RBIV-049L in the GenBank database (Accession number AY532606). The nucleotide sequence of RBIV-049L was predicted to encode an open reading frame of 142 amino acids, which
3.3. Expression and purification of recombinant protein GST-049L Small-scale cultures of the positive clones (selected on the basis of PCR screening) were subjected to IPTG induction to identify clones capable of expressing the predicted 42 kDa recombinant protein. The RBIV-049L gene was expressed in E. coli as a fusion protein with 26 kDa of glutathione Stransferase. From the analysis of SDS-PAGE, correct size recombinant protein was observed as a 42 kDa fusion protein
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Fig. 2. Detection of the target gene isolated from RBIV by PCR (A) and construction of recombinant plasmid pGEX-049L (B). (A) M1: 100 bp DNA ladder, lane 1: ORF 049L gene from RBIV; (B) M1: 100 bp DNA ladder, lane 1: pGEX-049L digested with EcoRI, lane 2: pGEX-049L digested with SalI, lane 3: pGEX-049L digested with EcoRI and SalI, M2: 1 kb DNA ladder.
(Fig. 5: panel A, lane 5). The results confirmed that the ORF049L was selectively expressed in the transformed E. coli BL21 (DE3) cells. Protein expression was not observed in the negative control, E. coli BL21 (DE3) cells, E. coli BL21 (DE3) cells transformed with the pGEX-4T-1 (induction), and E. coli BL21 (DE3) cells transformed with the pGEX-049L (without induction). The expression level of GST-049L fusion protein increased with the induction time. The highest amount of fusion protein was produced at 4 h after 0.4 mM IPTG induction. The soluble GST-049L fusion protein was applied to GSTrapFF affinity column chromatography, the band of GST-049L fusion protein showed high purities and correct size by SDS-PAGE (Fig. 5: panel A, lane 8).
adjuvant. Western blot was carried out to identify the reaction abilities of anti-RBIV-049L polyclonal antibody to recombinant RBIV-049L. Recombinant RBIV-049L protein could be detect by anti-RBIV-049L polyclonal antibody. (Fig. 5: panel B). Also, ELISA was performed to determine the proper dilution of antiserum against GST-049L. Anti-RBIV-049L polyclonal antibody could be detectable with 800 times dilution of the original polyclonal antibody solution (Fig. 6). However, 200 dilution was used for the detection of RBIV-049L for safety from the error.
3.4. Production of RBIV-049L-specific polyclonal antibody
RBIV in membrane fraction 1 from RBIV-infected rock breams (Fig. 7(A) lane 7, IM 1) could be detected by dot blot analysis using antiserum to GST-049L. The strongest reaction was observed in positive lane 1 of GST-049L. Strong signals were also observed in lane 5, 7, and 8 of total protein and membrane fraction 1 and 2 from RBIV-infected rock breams, respectively. Also, one-step PCR was performed to compare the sensitivity of dot blot analysis (Fig. 7(B)). Thick bands were observed in lane 1, 3, and 4 of DNA samples extracted from virus total protein and membrane fraction 1 and 2. To investigate the specificity and sensitivity of anti-RBIV049L serum against RBIV, the indirect ELISA assay was performed in total, cytosol and membrane fractions from RBIVinfected rock breams. As shown in Fig. 8, the indirect ELISA using anti-RBIV-049L serum recognized the total protein and membrane fraction 1 from RBIV-infected rock breams. Recent reports have suggested that direct PCR and one-step PCR (including two-step PCR) be used as sensitive and specific alternative protocols to western blot assay for the detection of fish viruses without sacrificing the animals [25,26]. Immunoblot detection system and one-step PCR combination-detection system would be useful to investigate the specificities and sensitivities of antibodies against fish viruses
BALB/c mice were immunized by an intraperitoneal injection with 100 mg of purified recombinant GST-049L emulsified with an equal volume of Freund’s complete
Fig. 3. Predicted membrane topology of RBIV-049L protein. Transmembrane helix predictions were performed with the computational method using the TMHMM Server v. 2.0. The graphs show the probability of a given amino acid residue of RBIV-049L as a transmembrane helix. The expected number of amino acids in transmembrane helices is 21.5. If this number is larger than 18 it is very likely to be a transmembrane protein (http://www.cbs.dtu.dk/services/ TMHMM-2.0/).
3.5. Detection of RBIV-049L from RBIV-infected rock breams
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Fig. 4. Comparisons of the nucleotides and amino acids sequences of RBIV-ORF049L to those of RBIV-049L-like ORFs of other Megalocytivirus species. (A) Alignment of the nucleotide sequences of PCR product from RBIV and other Megalocytivirus species. (B) Comparison of the amino acid sequence of RBIVORF049L to that of Orange-spotted grouper iridovirus ORF052L and infectious spleen and kidney necrosis virus ORF050L. Identical amino acid residues are darkly shaded, similar amino acids are lightly shaded. The alignment was created using the BioEdit Sequence Alignment Editor version 5.0.9 using the Clustal W algorithm. GenBank Accession numbers: Rock bream iridovirus (Rock bream iridovirus strain RBIV-KOR-TY1 from South Korea), AY532606; Orange-spotted grouper iridovirus, AY894343; infectious spleen and kidney necrosis virus, AF371960.
Fig. 5. Expression of fusion GST-049L protein in E. coli BL21 (DE3) and western blotting. (A) M: low molecular marker, lane 1: total protein of E. coli BL21 (DE3), lane 2: total protein of non-induced pGEX-4T-1, lane 3: total protein of induced pGEX-4T-1, lane 4: total protein of non-induced pGEX-049L, lane 5: total protein of induced pGEX-049L, lane 6: soluble protein of induced pGEX-049L, lane 7: insoluble protein of induced pGEX-049L, lane 8: purified protein of pGEX-049L (B) M: mid-range prestained protein size marker, lane 1: expressed GST-049L protein reacted with non-immunized mice serum (negative control), lane 2: non-induced GST049L protein reacted with anti-RBIV-049L polyclonal antibody (negative control), lane 3: expressed protein of GST-049L protein reacted with anti-RBIV-049L polyclonal antibody.
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Fig. 6. Determination of anti-RBIV-049L serum dilution concentration for indirect ELISA for RBIV detection.
Fig. 8. Detection of RBIV by indirect ELISA from virus-infected rock breams.
[27,28]. Considering the results, it could be suggested that dot blot detection system, indirect ELISA and one-step PCR combination-detection system be useful method for sensitive and specific detection of RBIV. Subcellular localization of virus in RBIV-infected rock breams evaluated by dot blot analysis,
indirect ELISA and one-step PCR detection system (Figs. 7 and 8). Direct amplification (direct PCR) from serum or blood samples using ORF 049L primers showed lower sensitivity than those of one-step PCR. Membrane fraction 1 (Fig. 7(A) lane 7, IM 1) from RBIVinfected rock breams showed strongest signals in the combination-detection system and the ORF 049L was located within the membrane fraction 1 from RBIV-infected rock breams. 4. Conclusions
Fig. 7. Sensitivity of the anti-RBIV-049L in dot blot analysis compared with one-step PCR 049L from RBIV-infected rock breams. (A) lane 1 (GST-049L): positive control, purified protein of pGEX-049L, lane 2 (NT): total protein of normal (uninfected) rock bream, lane 3 (NC): cytosol of normal rock bream, lane 4 (NM): membrane extracts of normal rock bream, lane 5 (IT): total protein of RBIV-infected rock bream, lane 6 (IC): cytosol of RBIV-infected rock bream, lane 7 (IM 1): membrane extract 1 (virus band) of RBIV-infected rock bream, lane 8 (IM 2): membrane extract of RBIV-infected rock bream, lane 9 (IM 3): membrane extract of RBIV-infected rock bream: lane 10 (BSA): negative control, lane A–F: total protein range from 300 to 0.003 mg (GST-049L: 3 mg to 0.03 ng). (B) One-step PCR of total genomic DNA from total (IT), cytosol extract (IC) and membrane extracts (IM 1, IM 2, IM 3) from the virusinfected rock bream.
The RBIV-specific polyclonal antibody was produced and characterized in this study. This paper is the first to report the overexpression and purification of the novel 16 kDa molecular protein, ORF 049L of RBIV, which is expected as a transmembrane protein of RBIV. The pGEX-4T-1 expression system was used for overexpression of RBIV-049L. The RBIV specific polyclonal antibody was produced using recombinant ORF 049L protein in mouse. Western blot was carried out to evaluate the reaction abilities of anti-RBIV-049L polyclonal antibody to GST-049L. Sensitivity and specificity of the antiRBIV-049L polyclonal antibody for the detection of RBIV antigens were improved by combination-detection system using dot blot analysis, indirect ELISA, and one-step PCR. ORF 049L located within the membrane fraction 1 from RBIVinfected rock breams. Furthermore, one-step PCR with dot blotindirect ELISA combination-detection system using ORF 049L and its antiserum showed sensitivity and specificity for the detection of RBIV. Our present results indicate that it can be possible to develop an expressed gene-based specific therapy against RBIV. Further studies are carried out for the evaluation of structure and function of the RBIV-049L protein. Acknowledgments This research was supported by Regional Research Fund from the Ministry of Commerce, Industry and Energy. Yun-Im
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