2.1. The recombinant plasmid
The gene sequence of HBP2 (Female-specific Histamine-Binding Protein 2, Rhipicephalus appendiculatus, Accesion number U96081) is deposited in the GenBank (NCBI, USA) with accession No. U96081, and it was obtained by requesting gene synthesis from Bioneer (Daejeon, Korea). The gene sequences of Cwp2p (Cell wall protein, the major mannoprotein of the yeast cell wall) is deposited in the GenBank with accession No. NM_001180025, and it was provided by Chungbuk National University (Lee et al. 2017).
The recombinant plasmid was constructed based on the pYES2 vector (Invitrogen) and was synthesized in the order of the Cwp2p signal peptide (60 bp), HA tag (27 bp) and histamine binding protein 2 (513 bp), Gly-Ser linker (30 bp) and Cwp2p gene (219 bp). The signal peptide and the stop codon of the histamine binding protein were excluded from the recombinant plasmid. The synthesized gene from HBP2 was provided in a pBHA vector containing the antibiotic Ampicillin resistance gene. After confirming the concentration of the vector, it was transformed into Escherichia coli DH5α to amplify the gene. E. coli DH5α cells were provided by the Real Biotech Corporation (Taipei, Taiwan).
2.2. Culture of recombinant E. coli & Plasmid isolation
Since the E. coli transformed with the synthesized vector contains resistance to ampicillin, it can be selectively cultured using medium containing ampicillin. For amplification of the vector, recombinant E. coli was cultured in Luria-Bertani medium (1% tryptone, 0.5% yeast extract, 1% NaCl and sterilization at 121 °C for 15 minutes) containing 100 μg/ml ampicillin. E. coli was incubated overnight at 37 °C at 180 rpm. After culturing, the cells were collected via centrifugation. The plasmids were extracted from the collected cells using Gene all Explin Plasmid SV kit that was provided by GeneAll Biotechnology (Seoul, Korea). The extracted plasmid was identified and was quantified via agarose gel electrophoresis.
2.3. Construction of plasmid DNA pYES2::CSP::HBP2::Cwp2p
The gene was cut with restriction enzymes and was ligated to insert DNA and vector DNA using the T4 ligase. The restriction enzymes and T4 ligase were provided by Enzynomics (Daejeon, Korea). The completed plasmid was precipitated via ethanol precipitation and was transformed into E. coli for amplification. The completed recombinant E. coli was incubated overnight at 180 rpm at 37 °C, and the plasmid was extracted.
The completed plasmid was subjected to a sequencing analysis using two sequencing primers of HBP2 seq F-552 (5'-TGA GTA CGC TGT AGG TAG GG) and HBP2 seq R-658 (5'-ATG GCG GCA GCG GAT TCA GA-3 '). Sequence analyses were performed via Cosmo Genetech (Daejeon, Korea).
2.4. Transformation in yeast S.cerevisiae
The completed plasmid was subjected to yeast transformation using the lithium acetate method (Schiestl and Gietz 1989), and the S. cerevisiae 2805 strain (ATCC 208280) was used for yeast transformation. S. cerevisiae strains were grown in Yeast Extract-Peptone-Dextrose (YPD) medium (1% yeast extract, 2% peptone and 2% D-glucose) until they had an optical density (OD) 600 of 0.9 – 1.0, and 1.5 ml of the strains were collected via centrifugation. Next, 1 ml of 0.2 M lithium acetate was added and washed. Then, the cells were collected via centrifugation again. 20 μL of 1 M lituuim acetate , 10 μL of plasmid and 30 μL of 70% polyethylene glycol were added to the collected cells, followed by incubation for 1 hour at 30 °C and 180 rpm. Next, Distilled water (DW) 140 μL was added and 100 μL cells were spread on synthetic dextrose (SD) solid medium (0.67% yeast nitrogen base, 0.5% casamino acid, 2% D-glucose, and 1.5% agar) to select transformed yeast.
The recombinant yeast in which HBP was expressed on the yeast surface was named GWL-1 cells (Cwp2p signal peptide :: HA tag :: HBP :: Gly-Ser linker :: Cwp2p :: pYES2). In addition, as a control, GWL-0 cells (Cwp2p signal peptide :: HA tag :: Gly-Ser linker :: Cwp2p :: pYES2) and Mock with only the pYES2 vector were transformed in the yeast.
2.5. Recombinant Yeast culture & induction
Mock and recombinant strains were each grown on SD minimal medium (0.67% yeast nitrogen base, 0.5% casamino acid and 2% D-glucose) until OD600 0.7 at 30 °C and 180 rpm. The grown cells were centrifuged at 3000 rpm for 5 minutes, and the supernatant was then removed. To induce the GAL promoter of pYES2 vector, the collected cells were suspended in synthetic galactose (SG) medium (0.67% yeast nitrogen base, 0.5% casamino acid and 2% D-galactose) and were cultured until OD600 0.78 at 30 °C for 20 hours at 180 rpm.
2.6. Histamine reduction ability test (LC-MS)
The cells were grown in SD medium to OD600 0.7 for the histamine inhibitory ability of the recombinant yeast. The cells were collected by centrifugation, and the medium was replaced by SG medium, followed by induction for 20 hours. The induced cells were cultured until OD600 0.78. The amount for 10 ml of induced cells was 1.30×107 CFU. Also, the cells were homogenized by a sonicator (SONICS & MATERIALS INC. 53 CHURCH HISLL RD. NEWTOWN, CT. U.S.A) and were tested to see if they could affect histamine even without living whole cells. Whole cells and cell debris were prepared by centrifugation and washing with DW. The whole cells and the cell debris were reacted with 1 ml of histamine solutions of various concentrations followed by a slow rotation for 2 hours. After the rotation, whole cells and cell debris were centrifuged, and the supernatant was filtrated to separate cells from the unreacted histamine solution. The histamine solutions for which the reaction was complete were quantitated via LC-MS analyses (Center for University-Wide Research Facilities, Jeonbuk National University, Korea).
2.7. Western blot
The cell debris was prepared by homogenizing the induced cells, and the protein lysate was prepared using NP-40 buffer (150 mM NaOH, 1.0% NP-40, 50 mM tris (pH 8.0)) containing protease and phosphatase. Their protein concentrations were quantitated using Quick Start Bradford 1X Dye reagent (Bio-rad). The samples were run on a 15% SDS-PAGE, transferred to nitrocellulose membranes, and were then blocked with 3% bovine serum albumin in T-TBS (20 mM Tris, 150 mM NaCl, 0.1% Tween 20 detergent). The membranes were incubated overnight at 4 ℃ with Anti-HA tag (abcam, 1:500 dilution). The blots were washed and incubated for 1 hour at room temperature with the Gt anti-Ms IgG (H+L) secondary antibodies (Invitrogen, 1:5000 dilution). Immunoreactive protein bands were visualized with ECL Prime Western Blotting Detection Reagents (Amersham) and light emission detected by exposing the membrane to X-ray film (AGFA).
The induced cells that were adjusted to OD600 0.5 were collected in 10 ml samples. The collected cells were washed 2 times using 1X Phosphate-buffered saline (PBS, NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 1.8 mM) containing protease, blocked 1 hour with 3% bovine serum albumin in 1X PBS, incubated overnight at 4 ℃ with Anti-HA tag (abcam, 1:500 dilution), washed 5 times using 3% BSA in 1X PBS containing protease, incubated for 1 hour at room temperature with m-IgG BP-FITC (Santa Cruz Biotechnology, 1:200 dilution) and washed using 1X PBS containing protease. The immunized cells were observed using an Axioscope (Carl Zeiss).
2.9. Animal cell culture
The murine macrophage RAW 264.7 cells were purchased from the Korean cell line bank (KCLB) and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO), supplemented with 10% Fetal Bovine Serum (GIBCO), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified 5% CO2 atmosphere at 37 °C.
2.10. MTT assay
The cytotoxicity of the histamine to RAW 264.7 cells was confirmed by an MTT assay. The cells adjusted to 1×105/ml were cultured for 24 hours and starved for 12 hours. The histamine concentration in normal human’s whole blood is 2.5 – 6.5 × 10-2 ppm (Jackson et al. 1998). Therefore, the histamine concentration used in this experiment was set to 10-2 – 10 ppm. The histamine solution was treated based on 1X PBS for 2 hours at 37 °C. MTT stock (50 mg/ml Thiazolyl Blue Tetrazolium Bromide) was diluted by free-serum DMEM and treated to RAW 264.7 cells for 2 hours. Dimethyl sulfoxide (DMSO) was treated and the absorbance was read at 540nm by a spectrophotometer. Then, the cell viability was calculated.
2.11. Phagocytosis assay
Phagocytosis assays were performed by a product manual provided by CELL BIOLABS. First, various concentrations of histamine solution were used that reacted and did not react with the induced GWL-1 cells. RAW 264.7 cells were adjusted to 5×105/ml, cultured for 24 hours, and starved for 12 hours. The histamine solution was prepared at 0.1, 1, 10 and 100 ppm based on 1X PBS. Then, the histamine solutions were reacted with GWL-1 cells 1.30×107 CFU/ml by a rotator for 2 hours and centrifuged, and then the supernatant was filtrated. RAW 264.7 cells were preincubated with a histamine solution that was diluted 1/10 with final concentrations of 0.01, 0.1, 1 and 10 ppm for 2 hours. Zymosan suspension provided by the phagocytosis assay kit was used as the phagocytosis stimulant. To measure the degree of phagocytosis, the absorbance was detected at 405 nm by a spectrophotometer, and the phagocytosis activity was calculated.
2.12. Data analysis
Each data point was obtained from three independent samples analyzed simultaneously to conduct error analysis. The averages are reported with the standard deviations and correlations for several experimental conditions. The data were analyzed using SigmaPlot (Systat Software, Inc., USA). A p-value < 0.05 was considered significant.