Chapter 2 Materials and methods
2.2 Methods
2.2.1 Cell culture condition
THP-1 were cultured in RPMI 1640 medium supplemented with 0.05 mM 2-mercantoethanol, 2 g/L sodium bicarbonate, 50 μg/ml of PSA, and 10% heat-inactivated fetal bovine serum (FBS). FO were cultured in DMEM medium supplemented with 1.5 g/L sodium biucarbonate, 10 % heat-inactivated FBS, and 50 μg/ml of PSA. PBMCs were cultured in RPMI medium supplemented with 1.5 g/L sodium biucarbonate, 10 % heat-inactivated FBS, and 50 μg/ml of PSA. Hybridoma were culture in DMEM medium supplemented with 1.5 g/L sodium biucarbonate, 20 % heat-inactivated FBS, and 50 μg/ml of PSA.
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2.2.2 Expression and purification of rHpHSP60 proteins Transformation of E.coli
E. coli [BL21(DE3)] (Yeastern Biotech, Taipei, Taiwan) were mixed
gently with 1 ng DNA then incubated on ice for 30 min. After incubation, cells were heat shock at 42℃ for 90 seconds followed by incubating on ice for 2 min. Next, placed the competent cells to 250 μl LB and incubated on 37C for 1 hour. Cells were plated on LB plate containing kanamycin (30 μg/ml) at 37C for 16 hours.Midi plasmid DNA preparation
Pick up a single colony was picked and inoculated in 100 ml LB medium containing kanamycin (30 μg/ml). After 16 hours incubation at 37C, the bacteria were recovered by centrifugation at 8000 rpm for 15 mins. Remove the supernatant as dry as possible, and then DNA was preparated by QIAamp DNA Midi kit according to the manifacture’s protocols. The precipitated DNA was washed by adding 1ml of 70%
ethanol, and then dissolved in DDW. Measure the absorbance at 260 and
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280 nm and assay by using restriction enzyme digestion for checking the DNA quantity and quality.
Expression and purification of rHpHSP60 protein
E. coli [BL21(DE3)] (Yeastern Biotech, Taipei, Taiwan) were
transformed with 1ng of DNA and grew on LB plate containing kanamycin (30 μg/ml) at 37C for 16 hours. Then single colony was picked and inoculated in 100 ml LB medium containing kanamycin (30μg/ml)). After 16 hours incubation at 37C, the bacteria in the LB broth were refreshed in 900 ml LB with vigorous shaking. Assayed the OD value until OD600 reaches 0.6~0.8, then protein induction was performed by adding 1.25 ml of IPTG (800mM) and continually incubated for 4 hours. After 4 hours incubation, harvested the bacteria by centrifugation at 5000 rpm for 15 min at 4 C. Discarded the supernatant and resuspended the pellet with 30 ml binding buffer containing urea (20 mM Na2HPO4, 0.5 M NaCl, 40 mM imidazole, 8 M urea, pH 7.4). Total lysates were sonicated for 15 min and then centrifuged at 12,000 rpm for 30 min to collect the supernatant which containing rHpHSP60.12
HisTrapHP column (General Electric, NY, USA) (1cm) was used to purify rHpHSP60. All the buffers and protein samples needed to be filtered with 0.45µm syringe filter. After protein sample loading into the column, washed the column with 30X volume of binding buffer to remove the unwanted proteins and then eluted the rHpHSP60 with 10X volume of elution buffer (20 mM Na2HPO4, 0.5 M NaCl, 500 mM imidazole, pH 7.4). Eluted rHpHSP60 were collected and loaded into G-25 column (General Electric, NY, USA) to remove the unnecessary salt and replace the buffer with PBS (Phosphate Buffer Saline, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KHPO4, pH 7.4). Protein concentration was quantified by Coomassie Plus reagent (BSA was used as the standard). SDS-PAGE and western blotting with anti-His conjugated HRP (ROCKLAND, PA, USA) were used to confirm the purity of rHpHSP60.
2.2.3 Peripheral blood mononuclear cells (PBMCs) isolation
PBMCs were isolated from human whole blood by using Ficoll-PaquePlus. Dilute human whole blood with equal volume of PBS.
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Added 6 ml Ficoll-PaquePlus into the 15 ml centrifuge tube and loaded 8 ml of the diluted blood sample on Ficoll-Paque Plus reagent carefully.
Then centrifuged the tubes at 400g for 40 min at 18C. After centrifugation, removed the plasma layer and collect the PBMCs carefully between the plasma layer and Ficoll-PaquePlus solution.
Washed the cell with 2X volume of PBS, centrifuged at 1,500 rpm for 15 min. Discarded the supernatant and added 10 ml of ACK lysis buffer (0.15M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) to lyse the red blood cells. Centrifuged at 1,500 rpm for 15 min to remove the supernatant and then washed the cell with 10ml PBS again. Counted the cell number and seeded the PBMCs into 96-well culture plate with RPMI1640 culture medium.
2.2.4 PBMC proliferation assay
To mimic the action occur when the immune cells encounter antigen, anti-CD3 antibody (kindly provided from Dr. Steve R. Roffler) (OKT3, an antibody that specifically binds to CD3 surface marker and transduces signals continually to activate T lymphocytes) was used to selectively
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activate T lymphocyte. Briefly, PBMCs were isolated from healthy donors then seeded into anti-CD3 mAb (0.03µg/well) pre-coated 96-well plates (2*105/well, triplicate for each treatment) and treated with different proteins (200 ng/ml) of HpHSP60, HpHSP60101-200, HpHSP601-200, HpHSP601-250, HpHSP60200-300, HpHSP60300-547 for four days incubation.
Cell proliferation was verified by MTT assay. In briefly, 96-well was centrifuged at 1,500 rpm for 15 min to remove the supernatant. Added 100 µl MTT solution into each well and incubated the plate at 37C for four hours. Then centrifuged again, removed the supernatant and dissolved the purple crystal with 100 µl DMSO each well. Shook the plate for 10 min then measured the OD value at 595nm. The proliferation index was calculated with following equation: proliferation = (OD value of protein-treated PBMC)
(OD value of PBMC treated without
HpHSP60)*100%.2.2.5 IL-8, IL-10, TNF-α, TGF-β secretion from PBMCc cells
PBMCs cells grown on 96 well plates were stimulated with 200 ng human HSP60, 200 ng HpHSP60, HpHSP60101-200, HpHSP601-200,
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HpHSP601-250, HpHSP60200-300, HpHSP60300-547 at 37℃ in RPMI medium for 2 days. Supernatants and cells were separated by centrifugation at 4000rpm for 5 minutes. Supernatants were assayed for IL-8, IL-10, TNF-α, TGF-β production by ELISA kit. (R&D system, MN, USA).
2.2.6 IL-8, IL-10, TNF-α, TGF-β secretion from THP-1 cells
THP-1 cells grown on 96 well plates were stimulated with 200 ng human HSP60, 200 ng HpHSP60, HpHSP60101-200, HpHSP601-200, HpHSP601-250, HpHSP60200-300, HpHSP60300-547 at 37℃ in THP-1 growth medium for 24 hours. Supernatants and cells were separated by centrifugation at 4000 rpm for 5 minutes. Supernatants were assayed for IL-8, IL-10, TNF-α, TGF-β production, and IL-8, IL-10, TNF-α, TGF-β was measured by IL-8, IL-10, TNF-α, TGF-β ELISA development kit.
(R&D system, MN, USA).
2.2.7 Production of monoclony antibody
Animal care and use
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The monoclony antibody productions were utilized Balb/c mice with 5-7 weeks of age. The mice were fed in animal room at Chiao Tung University during thr period of immunization. Feed and water were available daily.
Preparation of mouse polyclony antibodies against HpHSP60
Female Balb/c mice, aged 5-7 weeks, were used for immunization.
HpHSP60 in sterilized phosphate buffered saline (PBS), containing 0.12M NaCl, 0.02 M phosphate, pH 7.4, was mixed and homogenized with an equal volume of complete Freund’s adjuvant by a three-way stopcock. Each mouse was initially given a total emulsion of 0.3 ml containing 100μg of protein with 6 subcutaneous injections onto the back and an intraperitoneal injection. At day 7, an identical dose with incomplete was given intraperitoneally followed by two intramuscular injections without adjuvant at day 14. Seven days following a final booster, blood was collected in 0.1 % (wt/vol) EDTA and plasma was obtained. This plasma was used as a souerce for conventional polyclonal
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antibody against HpHSP60. Additional booster injections were given when necessary.
Preparation of mouse polyclony antibodies against HpHSP60
The spleen obtained was used for preparing hybridoma fusion after immunization. In brief, myeloma cell line (FO) was fused with spleen cells from immunized Balb/c mice at a ration of 1:5. Fusion was carried out within 2 min at 37℃ using 1 ml of 50% (wt/vol) polyethylene glycol containing 10 % (vol/vol) DMSO. Cell mixture was then washed and resuspended in HAT medium containing approximately 1×105 FO cells per ml. The suspended cells were distributed as 100 μl per well in 96-well plates and incubared at 37℃ in incubator followed by an addition of 100μl of fresh HAT medium after 7 days. Subsequently, culture medium was assayed for the production of specific antibodyes by ELISA. After primary screening, desired hybridomas were selected and recloned.
2.2.8 Statistical analysis
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The results are presented as mean ± SD. Significant is calculated by
T test. The P value < 0.05 is considered as statistically significant.19