Preparation of A. salmonicida
Atypical A. salmonicida (ace) (strain B10F21, T1031) was cultured on heart infusion agar (HI; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) plates for 72 h at 20 °C. The colonies were identified as ace using flow cytometry (FCM) using anti-A. salmonicida (anti-ace) monoclonal antibody (MAb)17. The selected colonies were sub-cultured in 500-ml HI broth for 72 h at 20°C. The concentration of bacteria was estimated by measuring the absorbance at 600 nm or CFU. Formalin-inactivation of the bacteria was conducted as described previously. Live and formalin-killed bacteria were used as antigens in the following experiments.
Cloning and construction of anti-A. salmonicida scFvs
The hybridoma clones producing MAbs against ace4A, and 8H, were established as previously described17. Total RNA from hybridoma cells was reverse transcribed using the SMART™ RACE cDNA amplification kit (Clontech, Mountain View, CA). PCR amplified cDNA fragments for the VH and VL regions with appropriate primers, and then mixed and assembled into the single-chain variable fragment (scFv)22.23 by four-step PCR amplification using appropriate primers containing linker sequence (Supplementary Figure S1 and Table S1). The resulting fragments were digested with NotI/xbaI and cloned into the pCAGGS-MCS expression vector24.25. The Myc-tag (EQKLISEEDL) was inserted into the xbaI/EcoRI site of all pCAG/anti-ace scFv constructs. The result was that all anti-ace-scFvs were fused with the Myc-tag at the C-terminus. The GenBank/EMBL/DDBJ accession numbers for the sequences of the cDNAs encoding VH and VL are 4A-VH, LC225752; 4A-VL, LC225755; 8H-VH, LC225753; 8H-VLLC225756.
Cells and electroporation
The murine T-cell hybridoma DO-11.1026 and hybridoma cells producing anti-ace antibodies were maintained in RPMI1640 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 4 mM L-glutamine, 10-mM HEPES (all from Life Technologies, Carlsbad, CA, USA), and 10% fetal calf serum. DO-11.10 cells adjusted to a concentration of 5 × 106 cells/400-μL culture medium with 1.25% dimethyl sulfoxide per cuvette were electroporated using a Gene Pulser (Bio-Rad, Hercules, CA, USA) with 20 μg plasmid DNA at 290-V and 960-μF.
Construction of plasmids for transgenic silkworms
The cDNA fragment for anti-ace-scFv-Myc was generated by PCR from pCAG/anti-ace-8H-scFv-Myc using the sense primer #13 and reverse primer #14 (Supplementary Table S1). The PCR product was digested with bamHI-SalI, inserted between the FibL promoter region through the FibL coding region (FibLpro-FibL) and FibL 3′-untranslated region (FibL-3′-UTR), and the fused DNA fragment of FibLpro-FibL-anti-ace -scFv-FibL-3′-UTR cloned into the AscI-ESFI site of pBac[3XP3-mKOafm] vector, which was replaced DsRed2 DNA sequence in the original vector, pBac[3XP3-DsRed2afm]27with monomeric Kusabira-Orange (mKO)28 as a selection marker. This construct was designated pBac[3XP3-mKOafm]-LC-anti- ace-scFv-Myc.
Generation of transgenic silkworm
Transgenic silkworms were generated as described elsewhere2 with minor modifications. The transgene plasmid and a helper plasmid vector, pHA3PIG, coding for piggyBac transposase18 were mixed at a concentration of 0.2 μg/μL each in 5 mM KCl and 0.5 mM phosphate buffer (pH 7.0) and injected into the fertilized eggs of the W/cs1 silkworm 5–10 h post-oviposition. The hatched larvae (G0) were reared on an artificial diet (Nihon Nosan, Kanagawa, Japan) at 25 °C until they developed into moths and were permitted to mate. Using fluorescent microscopy (MZ16FA, Leica Microsystems, Wetzlar, Germany), G1 embryos were screened for transgenic individuals with mKO expression 6–7 d after oviposition. Transgenic silkworms were reared and sib-mated for at least three generations. The experimental strain WS19 carried the transgene coding for the FibL fused with anti-ace-8H-scFv-Myc. Control strain S01 carried the transgene coding for the FibL fused with anti-WASP scFv-Myc10.
Solubilization of silk cocoons and preparation of silk solution
Three hundred milligrams of cocoon shells (2–3 shells) were chopped into 2–3-mm squares, washed in 5 mL 70% ethanol, and then dissolved in 3 mL 9 M LiBr and 90 mM Tris–HCl (pH 9.0). The cocoon squares were suspended for four hours at 37°C until the silk proteins had completely dissolved. The solubilized silk solutions were adjusted to a 10 mg/mL concentration in 2 M LiBr as a stock solution.
The gene-transfected DO-11.10 cells and silk solutions from W/cs1, S01, and WS19 strains were treated with 2 × SDS sample buffer, separated using 12% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Bio-Rad). The blots were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for one hour at room temperature and incubated with anti-Myc polyclonal antibody (MBL, code no. 562, Nagoya, Japan), anti-β-actin rabbit MAb ( Cell Signaling Technology, code no. #4970, Danvers, MA, USA), and anti-FibL polyclonal antibody (raised against a synthetic peptide representing FibL residues 67–80), followed by alkaline phosphatase-conjugated anti-rabbit immunoglobulins (Igs) (Dako, code no. D0306, Glostrup, Denmark). Immunoreactive proteins were detected using BCIP-NBT Solution Kit for Alkaline Phosphatase Stain (Nacalai Tesque).
The gene-transfected DO-11.10 cells were lysed using RIPA buffer (50 mM Tris–HCL pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail; Nacalai Tesque) on ice for one hour. Cell lysates were centrifuged at 10,000 × g for ten minutes at 4°C and the supernatants were used for ELISA. The stock silk solutions (10 mg/mL in 2 M LiBr) were diluted with 1 mM Tris–HCl (pH 8.0) to a concentration of 0.2 mg/mL. One hundred microliters of the cell lysate, culture supernatants from hybridoma cells, and diluted silk solutions were applied to 96 well plates and incubated overnight at 4°C. After washing thrice with PBS, each well was blocked using ELISA Diluent (BioLegend, San Diego, CA, USA) at room temperature for one hour. After five washes with PBS and Tween 20, formalin-inactivatedace (1.8×108 CFU/mL) was diluted, applied to the wells, and incubated at room temperature for 90 min. Binding was detected using sequential incubation of plates with anti- ace-4A MAb and HRP-conjugated anti-mouse IgG Fc (abcam, ab97265), followed by incubation with ELISA POD Substrate TMB solution (Nacalai Tesque). After color development, the reaction was stopped with 2N H2N/A4, and the absorbance was read at 450 nm using a microplate reader (iMark™ Microplate Reader; Bio-Rad).
Filtration assay using glass wool coated with affinity silk
One gram of glass wool (Masuda Corporation, Osaka, Japan) was soaked in a solution with or without 4 ml 0.2 mg/mL WS19, W/cs1, S01 in 1 mM Tris–HCl (pH 8.0), and was incubated at 4 ℃ overnight. After incubation, the glass wool was taken out and was washed with pure sterile water. This glass wool was then packed inside the commercial air-lift water filter (Suisaku eight-core mini; Suisaku Co., Ltd. Japan) (Fig. 3a). 4.9×107/mL CFU of liveace was suspended in 1.68 L distilled water and was equally divided into 12 beakers (140 mL/beaker). The filter units were inserted into each beaker, and the filtered bacterial solutions were collected at 0, 1, 3, 10, 30 min, 1, and 2 h after starting aeration. The turbidity of the obtained samples was measured using a spectrophotometer at 600 nm. Images of the filtered water were also taken at 0, 10, 30 min, 1, and 4 h of filtration. It was confirmed that the affinity silk was absorbed on the wool by Western blotting using anti-Myc-tag-pAb as the primary antibody and anti-IgG (H+L-chain) (Rabbit) pAb-HRP as secondary (MBL life science ) (data not shown).
The glass wool in the filtration units was ejected, thoroughly washed with 100 mL of distilled water in a beaker, and was treated with 2 mL 0.01% Triton X 100-PBS for ten minutes. The elute was obtained after centrifugation, diluted ten times in carbonate-bicarbonate buffer, and was subjected to an indirect ELISA. Anti- ace MAb 5H (1:5000 dilution of mouse ascites) and anti-IgG Fc Fragment antibody-HRP (MBL life science, Japan) of 1:50,000 dilution was used as the primary and secondary antibodies, respectively. Other steps in this ELISA were followed as described above. These experiments were repeated independently.
The Student’s t-test analyzed sample pairs. Multiple samples were evaluated using one-way ANOVA with Tukey’s test. Differences were considered significant whenp< 0.05.