2-1 Materials 2-1.1 Reagent
The following reagents obtained were described as following: RPMI 1640, Fetal Bovine Serum (FBS), BSA, and Tryzol were from Invitrogen Inc. (Gaithersburg, MD, USA).
Penicillin/ streptomycin/ amphotericin (PSA) were from Biological industries (Beithaemek, Israel). Restriction enzymes were from Promega Inc. (WI,USA). Kanamycin and Tris were from MDBio Inc. (Rockville, MD, USA). Ethidium bromide (EtBr), Isopropyl-beta- D-thiogalactopyranoside (IPTG), NaCl, yeast extract, agar, Tris-HCl, Triton X-100, TEMED and imidazole were from Amresco Inc. (Solon, OH, USA). Recombinant human TNF-α and TGF-β1 was from Peprotech Inc. (Rocky Hill, NJ). Sephadex G-25 Medium was from Amersham Bioscciences (Uppsala, Sweeden). Nitrocellulose (NC) paper was from PALL Inc.
(Ann Arbor, MI, USA).
2.1.2 Antibody
PE-conjugated anti-CD86, Fluorescent isothiocyanate (FITC)-conjugated anti-CD40, anti-CD80, anti-HLA-ABC, and anti-HLA-DR antibodies were from BioLegend (Sandiego, CA, USA). HRP-conjugated rabbit anti 6X His antibody was from Novus (Littleton, CO, USA).
2-1.3 Kit
Human IL-1β, IL-6, IL-8, and TNF-α ELISA kit was obtained from R&D systems
(Minneapolis, MN). Superscript III RT kit was from Invitrogen (Gaithersburg, MD, USA).
RealQ-PCR master mix kit was from Ampliqon (Copenhagen, Denmark). Coomasie PlusTM Protein Assay Reagent kit and Enhanced chemiluminescence (ECL) system was from Pierce (Rockford, IL, USA), FITC-dextran was from SIGMA-ALDRICH (Steinheim, Germany).
2.1.4 Instrument
HisTrapTM HP column was from GE healthcare (Uppsala, Sweeden). ABI PRISM 7000 from was Applied Biosystems (USA). Flow cytometer was from BD (Bedford, MA, USA). Human heat shock protein 60 cDNA (complementary DNA) library were kindly provided from Dr.
Chich-Sheng Lin (NCTU, Laboratory of Biomedical Engineering, Biological Science &
Technology Lab).
2.1.5 Bacteria
Escherichia coli (BL21 and DH5α) was from Yeastern Biotech Co. H. pylori genome
was from Department of Internal Medicine, College of Medicine, National Taiwan University.
2-1.6 Cell line
THP-1 cells, acute monocytic leukemia cell line was purchased from the Bioresource Collection and Research Centre (BCRC) (Hsinchu, Taiwan). Unlike other leukemic cell lines, THP-1 cells have no prominent chromosomal abnormalities (134).
2.2 Methods
2-2.1 Recombinant DNA techniques
The H. pylori strains were isolated from gastric biopsy specimens at National Taiwan University Hospital. The genome of H. pylori was prepared from the clinical isolates. The gene of Hsp60 was amplified from the genome of H. pylori by polymerase chain reaction (PCR) using the primers: 5’- ATC GAA TTC ATG GCA AAA GAA ATC AAA TTT TCA - 3’
as forward primer and 5’-GAT CTC GAG TTA CAT CAT GCC GCC CAT G-3’ as reverse primer. PCR condition was that 94 ℃ denaturation step followed by 35 cycles of 45 s at 95 ℃, 45 s at 50 ℃ and 2min at 72 ℃. After these cycles, incubate the PCR mixture at 72 ℃ 10min for complete elongation. PCR product was harvested, digested with EcoR1and Xho I, and inserted into EcoR I and Xho I restriction fragment of the expression vector pET-30a with N-terminal His-tags. The recombinant plasmids were further identified by restriction enzyme and agarose gel. The resulting plasmid pET- Hsp60 was transformed into competent E.coli BL21 (DE3) cells growing on an agar plate with kanamycin for selection.
2-2.2 Transformation
Remove the appropriate number of competent cells tubes from the -80 ℃ freezer. DH5α is used for cloning and DNA amplification. BL21 is used for protein expression. After the cells have thawed, add 1ng DNA into the cells, mix by gently swirling the tip or by gently
tapping the tube. Incubate the competent cell on ice for 30 min. Heat shock the cell at 42 ℃ for 90 s. Place the cells on ice for 2 min and add 250 μl LB (10g tryptone, 10g NaCl, 5g Yeast extract) and incubate at 37 ℃ with shaking 225 rpm for 1 hr. Spread 100μl mixture onto each LB agar plate (10g tryptone, 10g NaCl, 5g Yeast extract ,20g agar) containing kanamycin (30 mg/ml) and incubate at 37 ℃ for 12~16 hr.
2-2.3 Expression and purification of HpHsp60 gene in Escherichia coli
The colonies on the agar plate were picked, and shake in 100 ml LB with 30 μg/ml kanamycin at 37℃ overnight. Then, the 100 ml LB with bacteria was inoculated in 900 ml LB and the bacteria grew until the optical density at OD 600 nm reached 0.4-0.6. IPTG was added to a final concentration of 1 mM, and E. coli cells continually grew in 1L LB for 4 h.
After induction, the LB containing E. coli cells were harvested by centrifugation at 5000 rpm for 15 min and the pellet was resuspended in 30 ml binding buffer (20mM Na2HPO4, 0.5M NaCl, 40mM imidazole, pH7.4). Then the homogenized samples were sonicated with short burst of 1 sec followed by intervals 1 sec and the sonication processing was maintained for 15 min. Centrifuge the samples at 12000 rpm for 30 at 4 ℃. Harvest the supernatant and passed the 0.45 μM filter to remove the particles.
2-2.4 Protein purification
In this experiment, we purify our proteins with HisTrapTM HP column (GE healthcare).
To prepare the column, wash the column with 5 column volumes of DDW and equilibrate the
column with 5 column volume of binding buffer at the flow rate about 1 ml/min. Apply the pretreated sample and wash with wash buffer (20mM Na2HPO4, 0.5M NaCl, 60mM imidazole, pH7.4) about 60 volume. Elute with elution buffer (20mM Na2HPO4, 0.5M NaCl, 200mM imidazole, pH7.4, filtered with 0.45μm filter) for 10 volumes. Detect several fractions containing proteins by coomasie blue reagent. Collect the fractions with high protein concentrations and use G25 column to remove the unnecessary salt from the solution and replace the buffer with PBS (Phosphate Buffered Saline, 140 mM NaCl, 2.7mM KCl, 10 mM Na2HPO4, KH2PO4, pH 7.4). To prepare the G25 column, we need to swell the 7g Sephadex G-25 Medium with filtered PBS at room temperature for overnight. Fill the column with PBS and agitate the PBS containing G25 agarose, soon pour into the column along the edge of glass rod. After collecting the protein-containing fractions, we pour it into the G25 column and wash and elute with PBS. Detect which fractions contain proteins with coomasie blue reagent and collect the fractions. Poll the fractions together and filtered with 0.22 μm syringe filter. Quantitate the amount of protein concentration with coomasie blue reagent and dilute the solution to 1mg/ml. The quality of recombinant protein was checked by SDS-PAGE and Western blotting.
2-2.5 Cell cultures
The human monocyte THP-1 cell line was cultured according to therecommendations from ATCC. Briefly, non-adherent cells were grown in 75 Tflasks in RPMI 1640 culture
medium. The culture medium RPMI 1640 was supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 1% PSA (Biological Industris), 2.5 g/L glucose, 10 mM HEPES and 1.0 mM sodium pyruvate (from MP Biomedicals ) and 0.05 mM 2-mercaptoethanol (Amersco). Cells were incubated in tissue culture incubator with 5 % CO2 at 37 ℃ and split at a densityof ~5x105 cells/ml. Cells were kept no more than 2 months in culture from the
original stock.
2-2.6 Detection of cytokines production in the THP-1 cells using ELISA
THP-1 cells (5×105 cells per well) were dispensed into 24-well culture plates and then except for control treated with 10 μg/ml rHpHSP60. The supernatants with or without rHpHSP60 stimulation were collected at 24 h. To detect the kinetic of cytokine protein expression, the supernatants were collected at 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h and frozen at −80 ◦C. The concentration of TNF-α, IL-1β, IL-6, and IL-8 in the supernatants was measured by Enzyme-Linked ImmunoSorbent Assay (ELISA) kit (R&D systems).
2-2.7 FITC-dextran uptake of THP-1 cells
THP-1 cells were seeded in 24-well tissue culture plates at a density of 5×105/ml in a volume of 1 ml per well. Except for the control, 10 μg/ml of rHpHSP60 was added into the culture medium. To examine the effect of TNF-α or TNF-α combing with TGF-β1 on endocytotic activity of THP-1 cells, cells incubated with seven concentrations of TNF-α or 1 ng/ml TNF-α with three indicated concentration of TGF-β1. After 16 h treatment, cells with
culture medium were collected and centrifuged with 2000 rpm 5min. Then, the culture medium were removed and cells were incubated in 2% RPMI medium containing 1 mg/ml FITC–dextran in the tissue culture incubator with 5 % CO2 at 37 ℃ or upon the ice (as the control) for 2 h. The uptake of FITC–dextran was analyzed using flow cytomestry.
2-2.8 Surface marker detection on THP-1 cells
Cells treated with 10 μg/ml rHpHSP60 or with TNF-α combing with TGF-β1 for 16 h or with three concentrations of TNF-α for 24 h. Then, cells with culture medium were collected and centrifuged with 2000 rpm 5min. The cell pallet was washed by wash buffer once time.
Phosphate-buffered saline (PBS) with 1% bovine serum albumin and 0.05% sodium azide was used as wash buffer. Cells were incubated for 1 h on the ice at a volume of 50 λ with 1 λ of the following antibodies: PE-conjugated anti-CD86, Fluorescent isothiocyanate (FITC)-conjugated anti-CD40, anti-CD80, anti-HLA-ABC, or anti-HLA-DR antibodies. After three times washing, the surface marker expression of THP-1 cells was assayed by flow cytomestry.
2-2.9 RNA isolation and cDNA synthesis
At each time point, media was removed from the microcentrifuge tube after centrifugation. Total RNA was extracted from cells using Trizol reagent as described in the manufacturer’s protocol. After centrifugation, pellet cells lysed in 1 ml Trizol reagent by repetitive pipetting. Incubated the homogenized samples for 5 minutes at room temperature to
permit the complete dissociation of nucleoprotein complexes. Add 200 λ of chloroform per 1 ml Trizol reagent. Cap sample tubes securely. Shake tubes fiercely by hand for 15 seconds and incubated them at room temperature for 3 minutes. Centrifuge the samples at 12000 rpm for 15 min at 4 °C. Following centrifugation, transfer the colorless upper aqueous phase to a fresh tube. Precipitate the RNA from the aqueous phase by mixing with 500 λ isopropyl alcohol.
Incubated samples at room temperature for 10 minutes and centrifuged at 12000 rpm for 20 minutes at 4 ℃. After centrifugation, the gel-like pellet on the side and bottom of the tube.
Remove the supernatant. Wash the RNA pellet once with 1 ml of 75 % ethanol. Mix the samples by vortexing and centrifuge at 7500 rpm for 10 minutes at 4 ℃. Following centrifugation, remove the supernatant and briefly air-dry the RNA pellet for 15 minutes.
Dissolved RNA in RNase-free water and incubated for 10 minutes at 60 ℃ . The
concentration of RNA was measured by spectrophotometry (ideal OD 260/280 ≌ 1.8-2.0) and the samples were stored at -80 ℃ until use. One microgram of total RNA was used to synthesis cDNA using random hexamer primers with the Superscript III Fisrst-Strand Synthesis kit (Invitrogen).
2-2.10 Quantitative Real-time PCR
The resulting cDNA was then subjected to quantitative real-time PCR and primers used were as Table 1. cDNAs were amplified using SYBR®-PCR mastermix (Applied Bio-systems) according to the recommendations of the manufacturer in a total volume of 25 μl in a ABI PRISM 7000 system (Applied Biosystems). The reactions were incubated at 50
℃ for 2 min to activate uracil N-glycosylase and then for 4 min at 95 ℃ to inactivate this enzyme and activate the Amplitaq Gold polymerase, followed by 40 cycles of 15 sec denaturation at 95 ℃, 25 sec annealing at 60 ℃, and 25 sec extension at 72 ℃. The expression of each gene was normalized to β-actin, a housekeeping gene. All samples were run in duplicate and non-template control.