Cell strain, bacterial strain and plasmids
A human normal gastric epithelial cell line, GES-1, and a H. pylori reference strain (J99, ATCC 700824, cagA+/vacA s1m1) were provided by the Cancer Research Institute of Central South University, Changsha, China. Escherichia coli strain, TOP10, was purchased from Genscript Biotech (Nanjing) Corp Co., Ltd., Nanjing, China, and E. coli BL21(DE3) strain and plasmid pET41b were purchased from Novagen Company, Madison, USA.
Construction of a VacA recombinant expressing vector
Genomic DNA of H. pylori was extracted according to the standard protocol for MiniBEST Bacterial Genomic DNA Extraction Kit (Takara Biotechnology Co., Ltd). The vacA gene of H. pylori (strain J99 / ATCC 700824) was amplified from the sequences (mature protein fragment of VacA toxin, expressing amino acids 34 to 858) by polymerase chain reaction (PCR) method using the mutation primers and gene primers (sequence F1: 5’CAATCGTTGGCGGCATCGCTACGGGTACGGCTGTTGGCACGGTTTCGGGCCTGCTTAGTTGGGGACTC3’, sequence F:5’CTTTAAGAAGGAGATATACATATGTTTTTCACCACGGTTATCATTCCGGCAATCGTTGGCGGCATCGCT3’);F1 contained NdeI sites, and F contained XhoI sites. Conditions were as follows: Pre-denaturation at 96 °C for 5 min; and 25 cycles of 96 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min 20 s and 72 °C for 5 min. The PCR product was digested with NdeI and XhoI, and inserted into the expression vector plasmid pET41b containing C-terminal histidine tag (8His.tag; Novagen Company). The PCR product and the inserted pET41b plasmid were then combined at a 10: 1 M ratio in a ligation reaction containing 1× ligation buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM ATP, 1 mM DL-dithiothreitol (DTT), 25% (w/v) polyethylene glycol 8000) with five units of T4 DNA ligase (Gibco BRL, USA) to reach a final volume of 20 μL and incubated overnight at 16 °C. The combination product (10 μL, 300 ng) was mixed with competent cells (100 μL, 1 × 108 cfu/μg, the optical density of the cells at 600 nm reached 0.5–0.8) of E.coli TOP10 on ice for 30 min, in heat shock at 42 °C for 60 s, and then instantly incubated in an ice bath for 2 min. The solution was grown in 800 μL Luria-Bertani (LB) medium, preheated to room temperature, on a shaking incubator for 1.5 h at 37 °C. Afterwards, the cells were harvested by centrifugation at 13,523 g for 2 min and removal of 200 μL supernatant. The solution was resuspended with 200 μL LB medium containing 50 μg/mL kanamycin and then the LB medium was evenly spread into a solid LB medium plate. Following this, the plate was reversely placed and cultured at 37 °C overnight. Five well-grown colonies on the solid LB medium plate were selected and inoculated into 5 mL LB medium containing 50 μg/mL kanamycin. Transformants were grown at 37 °C overnight with shaking. Plasmids were extracted by the methods described by Green et al. . Afterwards, the plasmids were digested with NdeI and XhoI. The restricted product, a 2502-bp positive clone, was assessed by 1% agarose gel electrophoresis. The correct recombinant clones were then validated by Sanger dideoxy sequencing, with the Applied Biosystems 3730XL DNA Analyzer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Expression and purification of VacA recombinant protein
Expression of VacA recombinant protein
Recombinant plasmid containing the vacA insert (pET41b-vacA34–854) was transformed into E. coli BL21(DE3) (Novagen Company). Briefly, E. coli cells were incubated in 50 mL LB medium containing 50 μg/mL kanamycin, and incubated overnight at 37 °C with shaking at 225 g. Then, the 10-mL pre-culture above was seeded into 20 × 500 mL Terrific Broth containing 50 μg/mL kanamycin in Erlenmeyer flasks, and incubated at 37 °C with shaking at 225 g. When the OD600 value of the culture reached 1.2, IPTG was introduced at the final concentration of 0.5 mM into the culture to induce the protein expression at 15 °C for 16 h with shaking at 225 g. The cell pellet was harvested at 8000 g, maintained at 4 °C for 20 min. Following this, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis were used to detect the VacA protein in E.coli expression in construct pET41b.
Purification of VacA recombinant protein
The cell pellet was harvested at 8000 g, at 4 °C for 20 min, and re-suspended with the lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). The cells were lysed using a sonicator (protocol: 3 s on and 6 s off cycles for a total of 15 min at 500 w) and the cell lysate was centrifuged at 14,650 g, at 4 °C for 30 min. The supernatant and inclusion bodies were separately collected for the purification of the VacA recombinant protein as described below.
Purification of VacA recombinant protein from the supernatant
The target protein was purified from the supernatant with Ni-IDA (Ni-IDA resin, Genscript Biotech (Nanjing) Corp Co., Ltd). The above-mentioned lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) was used as the column equilibration buffer. The target protein was eluted with a stepwise gradient of imidazole (50 mM Tris-HCl, 150 mM NaCl, pH 8.0 + a gradient concentration of imidazole, i.e. 20, 50, 100 and 500 mM) and then washed with washing buffer (50 mM Tris, 150 mM NaCl, 1% TritonX-114, pH 8.0. The 1% TritonX-114 acts to remove the endotoxin). Western blotting and SDS-PAGE were used to analyze the purification process. According to the results of Western blotting and SDS-PAGE, the target proteins in the lane with the most protein expression were pooled and dialyzed into buffer 1 × PBS, pH 7.4. The dialysis was performed in a 14 kDa cut-off dialysis membrane (VISKASE® Companies, Inc.) for 4 h and the above buffer was replaced with the same fresh buffer (1 × PBS, pH 7.4) for an additional 16 h. After dialysis, the sample was centrifuged at 14,650 g for 30 min and filtered through a 0.22 μm filter (Merck Millipore). The final quality control (QC) included SDS-PAGE along with western blotting.
Purification of VacA recombinant protein from inclusion body
For the purification of VacA recombinant protein in the inclusion bodies, the inclusion body pellet was solubilized in the denature buffer (50 mM Tris-HCl, 8 M Urea, pH 8.0) by sonication. The cell precipitate was centrifuged at 14,650 g for 30 min at 4 °C, and the supernatant was used for further purification. Ni-NTA (Profinity IMAC Ni-Charged Resin, Bio-Rad Laboratories, Inc.) affinity chromatography was applied to collect the recombinant protein in the supernatant. The above-mentioned denature buffer was used as the column equilibration buffer, and the target protein was eluted with a stepwise gradient of imidazole (50 mM Tris-HCl, 8 M urea, pH 8.0 + a gradient concentration imidazole, i.e. 20, 500 mM) after being washed with washing buffer (50 mM Tris-HCl, 1% Triton-X114, 8 M urea, 150 mM NaCl, PH 8.0). SDS-PAGE was used to analyze the fractions in the purification process.
Refolding, preservation and identification of VacA recombinant protein
Refolding and preservation of VacA recombinant protein
The target protein eluted in the above inclusion body purification was pooled, and a small-scale refolding test was performed using the following refolding buffers and methods: buffer 1: 50 mM Tris-HCL, 10% Gly, 150 mM NaCl, pH 8; buffer 2: 50 mM Tris-HCL, 10% Gly, 150 mM NaCl, 0.1 mM DTT, pH 8.0; buffer 3: 1 × PBS pH 7.4; buffer 4: 1 × PBS, 10% GLy, 500 mM NaCl, pH 7.4; buffer 5: 1 × PBS, 10% GLy, 500 mM NaCl, 0.1 mM DTT, pH 7.4; buffer 6: 20 mM Tris-HCL, 2 M urea, 400 mM Arg, 2.5 mM cysteamine, 0.25 mM cystamine, pH 8.5; buffer 7: 50 mM acetic acid, pH 2.9. Analysis of protein refolding was based on the protein solubility (when the protein was dissolved in the buffer, the appearance of the visible precipitate indicated refolding failure, whereas the complete dissolution indicated successful refolding), or the results of SDS-PAGE and western blotting (the lysate was collected and the protein content was further analyzed by SDS-PAGE and western blotting). The dialysis was performed in 14 kDa cut-off dialysis membrane for 4 h and for additional 16 h after replacement of the fresh buffer (i.e. the corresponding dialysis buffer in the dialysis method). Then, dialysis was performed in the final buffer (i.e. the corresponding dialysis buffer in the dialysis method) for 16 h. The most suitable protein final solution was selected based on protein refolding results as well as the buffer solvent used in the solution, which must not interfere with the biological activity detection of the target protein.
Identification of VacA recombinant protein
After dialysis, the sample was centrifuged at 14,650 g for 30 min and filtered through a 0.22 μm filter. The final QC included SDS-PAGE along with western blotting.
The protein sample was added to the loading buffer (300 mM Tris-HCl, 10% SDS, 30% glycerol, 0.05% bromophenol blue, 250 mM DTT, pH 6.8). The mixture was vortexed for 1 min, heated at 100 °C for 10 min, and centrifuged at 11,270 g for 1 min. The supernatant was taken for SDS-PAGE analysis (Gel: 4% ~ 20% gradient SDS-PAGE gel, Genscript Biotech (Nanjing) Corp Co., Ltd). Equal amounts (2 μg) of bovine serum albumin, (BSA, 67 kDa) and the protein were added to the loading buffer. The next steps were performed as described above. After the electrophoresis was completed, the band of the target protein was compared with the band of the reference protein (BSA), and the amount of the target protein was initially obtained. PAGE-MASTER Protein Standard (Genscript Biotech (Nanjing) Corp Co., Ltd) was used as a protein molecular weight marker.
Western blotting analysis
After SDS-PAGE, the precast gel (4% ~ 20% gradient SDS-PAGE gel, Genscript Biotech (Nanjing) Corp Co., Ltd) was fixed on electro phoretic apparatus and the inside of the electrophoresis tank was topped up with MOPS electrophoresis buffer. Then, the protein samples were added into the gel holes. Easy Western Protein Standard (Genscript Biotech (Nanjing) Corp Co., Ltd) was used as a protein molecular weight marker. SDS-PAGE was run at 140 V for 60 min, and stopped when the bromophenol blue reached the bottom of the separation gel. The gel was removed, and transferred onto the PVDF membrane (Bio-Rad Laboratories, Inc.) by using e-blot (a highly efficient wet protein transfer system). The membrane was washed with 1 × PBST (1× phosphate buffered saline (PBS), 0.06% Tween-20, pH 7.4) buffer twice, for 5 min each time, removed and sealed with rapid sealing liquid (Genscript Biotech (Nanjing) Corp Co., Ltd) for 8 min. The membrane was washed with 1 × PBST buffer twice, for 5 min each time, and then incubated at 4 °C overnight with skin milk containing the primary antibody (anti-His antibody, 4000:1). Then, the membrane was washed again, as described above, and incubated with skin milk containing the second antibody (anti-mouse antibody, 5000:1) for 45 min. Finally, the membrane was washed, as described above, and exposed to X-ray film. The bands were visualized using ECL Western Blotting Substrates (Promega Biotech Co., Ltd).
Detection of apoptotic activity of VacA recombinant protein
In a pilot experiment, we started with concentration 5μg/ml, gradually increased the protein concentration, incubated VacA protein with different concentrations and gradients and observed that cell morphological changes at each time point. It was found that apoptosis could be changed after the protein was incubated for a long time (48 h) with a low concentration (5μg/ml) or stimulated for a short time (24 h) with a high concentration (65μg/ml), and the effect of high concentration was more significant. Finally, we used a higher concentration (65μg/ml) for further experiments to determine the apoptotic activity of VacA recombinant protein as described below.
The morphological observation
GES-1 cells were cultured in RPMI 1640 medium (HyClone; GE Healthcare Life Sciences) containing 10% fetal bovine serum (Biological Industries Israel Beit-Haemek, Ltd) that was replaced daily, with trypsinization. Well-grown GES-1 cells were seeded into each well of a 6-well plate, containing 5 × 106 cells, and incubated with VacA recombinant protein (with a final concentration of 65 μg/mL in the experimental group) in an incubator at 37 °C in an atmosphere of 5% CO2 for 24 h. At the same time, other batches of GES-1 cells were incubated with RPMI 1640 medium and incubated with isovolumetric protein buffers (buffer 7). These cells served as a blank control group and buffer control group. The plate was removed from the incubator at a later point. Cellular structural changes, including nuclear cytoplasmic apoptosis and cytoplasmic vacuolization, were visualized before incubation and after 6, 12 and 24 h of incubation by invert microscopy. After incubation for 24 h, a single cell suspension was prepared by trypsinization and the samples were made into pathological sections according to conventional methods and examined by transmission electron microscopy, as previously described .
The terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay
Analysis of apoptotic cells was performed using TUNEL assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. GES-1 cells grown on glass coverslips in 6-well plates were treated with VacA recombinant protein (with a final concentration of 65 μg/mL in the experimental group) as described above, after incubation for 24 h, cells were then washed with PBS, fixed with 4% paraformaldehyde in PBS at room temperature for 30 min, and washed once with PBS. Then, cells were permeabilized in 0.5% Triton X-100 for 5 min and washed twice with PBS. The cells in each well were then incubated with 100 μL of TUNEL reaction mixture containing TdT and Cyanine 3-dUTP at 37 °C in the dark for 1 h. After incubation, cells were washed three times with PBS and mounted in antifade mounting medium containing DAPI. The Cyanine 3-labeled TUNEL-positive cells were captured using a fluorescent microscopy at 400× magnification by using 550 nm excitation and 570 nm emission (red fluorescence). The nuclei were counterstained with DAPI, and the TUNEL-positive cells with red fluorescent staining, indicative of apoptosis. Cells from 20 images for each sample were counted and the percent of apoptotic cells was calculated by dividing the number of apoptotic cells by the number of total cells counted. All assays were performed in triplicate.
All the data were expressed as mean ± standard deviation (SD) and the statistical analysis was performed using SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA), with Tukey-Kramer multiple comparison procedure, was used to determine the significance of differences among groups, Statistical significance was set at P < 0.05 .