modular architecture research tool (SMART) version 4.0 and the conserved domain search program of NCBI. The molecular mass and theoretical isoelectric point (pI) were predicted using EditSeq in DNASTAR software package (DNASTAR Inc. Madison, WI, USA). Multiple sequence alignment was performed with the ClustalX program. Signal peptide search was performed with SignalP 3.0. Plasmid construction and mutation of CsISG15 To construct pEtCsISG15, which expresses a His-tagged CsISG15, the coding sequence of CsISG15 was amplified by PCR with primers CsISGF1 (59- GATATCATGGAAATAAC- CATCACAA -39; underlined sequence, EcoRV site) and CsISGR1 (59- GATATCGCCAATGACGGTGT -39; underlined sequence, EcoRV site); the PCR products were ligated with the T– A cloning vector pBS-T (Tiangen, Beijing, China), and the recombinant plasmid was digested with EcoRV to retrieve the CsISG15-containing fragment, which was inserted into pET259 [35] at the EcoRV site. To construct pEtCsISG15M, which expresses a mutant CsISG15, CsISG15M, bearing alanine substitutions at R153 and G154 (so that the conserved LRGG motif was mutated to LAAG), a two-step PCR was carried out as follows: the first PCR was performed with primers CsISGF1 and MR3 (59 - GCCAATGACGGTGTGTCCTCTGCCTGCTGC- CAGACGCAGT -39); the PCR products were diluted 1006, and the dilution was used as a temperate for the second PCR with primers CsISGF1 and CsISGR1. The PCR products were inserted into pET259 at the EcoRV site as described above, resulting in pEtCsISG15M. Purification of recombinant protein and preparation of antiserum Escherichia coli Rosetta (DE3) (Bioteke, Beijing, China) was transformed with pEtCsISG15, pEtCsISG15M, and the empty vector pET259, resulting in transformants DE3/pEtCsISG15, DE3/pEtCsISG15M, and DE3/pET259 respectively. The trans- formants were cultured in Luria-Bertani broth (LB) medium at 37uC to mid-log phase, and isopropyl-b-D-thiogalactopyranoside was added to the culture to a final concentration of 1 mM. After growth at 18uC for an additional 10 h, recombinant protein was purified using nickel-nitrilotriacetic acid columns (GE Healthcare, USA) as recommended by the manufacturer. Purified proteins and the preparation from DE3/pET259 (named E. coli preparation) were dialyzed for 24 h against phosphate-buffered saline (PBS) and concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore, Billerica, MA, USA). The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and visualized after staining with Coomassie brilliant blue R-250 (Figure S1). The concentrations of the purified proteins were determined using the Bradford method with bovine serum albumin as a standard. To prepare anti-rCsISG15 antibodies, adult rats (purchased from the Institute for Drug Control, Qingdao, China) were immunized via subcutaneous injection with rCsISG15 mixed in complete Freund’s adjuvant. The rats were boosted at 20, 32, and 45 days after the initial immunization. The rats were bled 14 days after the last boost, and sera were obtained from the blood. The titer and specificity of the serum antibodies were determined by enzyme-linked immunosorbent assay and Western immunoblot analysis as described previously [36]. Western immunoblot analysis showed that anti- rCsISG15 serum could bind to rCsISG15 and rCsISG15M but not to purified rCsFerM, a recombinant ferritin of tongue sole [34] (Figure S2 and data not shown). Cytokine Property of Tongue Sole ISG15 CsISG15 expression in fish tissues (i) CsISG15 expression under normal physiological conditions. Brain, muscle, heart, gut, kidney, spleen, gill, and liver were taken aseptically from five fish (,12.7 g) and used for total RNA extraction with the RNAprep Tissue Kit (Tiangen, Beijing, China). One microgram of total RNA was used for cDNA synthesis with the Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real time reverse transcriptase- PCR (qRT-PCR) was carried out in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) using the SYBR ExScript qRT- PCR Kit (Takara, Dalian, China) as described previously [37]. PCR efficiency (99.9%) was determined as described previously [38]. Melting curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The expression level of CsISG15 was analyzed using comparative threshold cycle method (22DDCT) with b-actin as a control. The primers used to PCR the b-actin gene were reported previously [39]. The primers used to PCR CsISG15 were CsISG15RTF1 (59 –CTACGGTCTGCG- TTCTGGC -39) and CsISG15RTR1 (59 –CACCCTGCGCT- TAAAGTTGG -39). All PCR were performed in triplicate, and the data are given in terms of mRNA levels relative to that of b- actin and expressed as means plus or minus standard errors of the means (SE). (ii) CsISG15 expression in response to pathogen infection. The fish bacterial pathogen Vibrio anguillarum C312 [37] was cultured in LB medium and resuspended in PBS to 16107 colony forming units (CFU)/ml. Tongue sole (,12.7 g) were divided randomly into three groups (N = 30) and injected intraperitoneally with 100 ml PBS containing 106 CFU V. anguillarum or 104 copies of the megalocytivirus RBIV-C1 [40] or with 100 ml PBS alone (control). Fish (five for each time point) were sacrificed at 1 h, 4 h, 12 h, 24 h, and 48 h post-infection, and tissues were taken under aseptic conditions. For mRNA expression analysis, total RNA was extracted and used for qRT- PCR as described above. For Western blot analysis, proteins were extracted from 33 mg of tissues with the Sample Grinding Kit (GE Healthcare, New Jersey, USA), and equal volumes of protein preparations were loaded into SDS-PAGE gels. After electropho- resis, the proteins were transferred to a nitrocellulose membrane (Amersham, Cambridge, UK). Immunoblot was performed as described previously [41] using anti-rCsISG15 antibodies or anti-b-actin antibody (Bioss, Beijing, China). CsISG15 expression and localization in head kidney (HK) lymphocytes HK lymphocytes were prepared as described previously [42] and cultured in 96-well tissue culture plates containing L-15 medium (Thermo Scientific HyClone, Beijing, China) supple- mented with 10% calf serum. The culture was maintained at 25uC. To examine the effect of viral infection on CsISG15 expression, megalocytivirus RBIV-C1 was suspended in L-15 to 105 copies/ml. HK lymphocytes were washed and added with fresh L-15 containing or not containing megalocytivirus (104 copies/well). The cells were incubated at 25uC for 1 h, 2 h, 4 h, 8 h, and 16 h respectively and washed 36 with PBS. The cells were then examined for CsISG15 mRNA expression and extracellular production of CsISG15 protein. For mRNA expres- sion analysis, total RNA was prepared from the cells with Total RNA Kit I of Omega Bio-tek (Norcross, GA, USA) and used for qRT-PCR analysis of CsISG15 expression as described above. For extracellular protein production analysis, the culture supernatant of 56106 cells was collected by centrifugation and concentrated with Amicon Ultra Centrifugal Filter Devices (Millipore, Billerica, PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e44884