Plasmid and strain: Recombinant plasmid pET-32a containing the target gene, pro-B. fragilis enterotoxin-2 without the signal peptide nucleotide sequence (proBFT-2), and positive clones of Escherichia coli (E. coli) strain Rosetta (DE3) were provided by Shanghai Biotechnology Corporation (GenBank ID: AB026626).
Cell-line: The human colorectal adenocarcinoma cell-line SW-480 was purchased from Shanghai Cell Bank of the Chinese Academy of Science.
Animal experiments: Seventy SPF certified C57BL/6J mice with a 17-g body weight, aged 5 wk were purchased and fed in the Animal Center of the Institute of Biomedical Laboratory of the East China Normal University. The production license was SCXK (Shanghai) 2011-0031, and the usage license was SYXK (Shanghai) 2010-0094. The animal experiment was approved by the 1990 Animal Ethics Committee of the East China Normal University, and the controlled environmental factors for experimentation strictly followed the relevant regulations of the national standard GB14925-2010 of the experimental animals that were released and implemented by the National Administration of Quality Supervision and Quarantine. All animals were permitted free access to food and water.
Reagents: Ni-NTA agarose gels were purchased from GE (United States). DMEM medium was purchased from Invitrogen (United States). IPTG was purchased from Sigma (United States). TEV protease was provided by Bioengineering (Shanghai, China). Azomethane (AOM) and dextran sulfate sodium (DSS) that was used to induce a state of CRC in the experimental mice were purchased from Sigma and MP (United States), respectively. Ki-67 and Caspase-3 antibodies were purchased from Abcam (United Kingdom).
Expression and purification of recombinant protein proBFT-2: The nucleotide sequence coding for proBFT-2 without the signal peptide nucleotide sequence as the target gene was cloned to construct a recombinant plasmid pET-32a to produce the positive transformed clone strain DE3. Then, the positive transformed clone strains were cultured on a small scale, induced by IPTG, screened for the optimal condition of IPTG induction concerning concentration, temperature and time, and the SDS-PAGE method was utilized to select the best protein expression-inducing conditions. Then, the positive clones of DE3 with optimized inducing conditions were induced on a large scale. After IPTG induction of expression and cell sonication, the culture was centrifuged and the supernatants are applied to Ni-agarose affinity chromatography. The eluate was collected and tested by SDS-PAGE to detect recombinant proBFT-2 expression.
Analysis of the purified proBFT-2: ProBFT-2 was mixed with the TEV enzyme to remove the His-tag, and then an enzyme-digested protein solution was purified by nickel agarose affinity column chromatography. Finally, SDS-PAGE was used to detect the sample solution and elution before and after enzyme digestion.
ProBFT-2 digestion and hydrolytic release of BFT-2: In this procedure, 10 μg/mL trypsin was incubated with proBFT-2 solution at 37 °C for 1 h, leading to release of BFT-2 by proBFT-2 protein hydrolysis, following which it was purified by nickel column affinity chromatography, and the eluted fractions were collected. After the elution was dialyzed and purified, SDS-PAGE was performed. Finally, the elution was preserved at -80 °C.
Detection of BFT-2 biological activity: During the log phase of growth, 5 × 105 SW-480 cells were seeded into a 24-well plate with a total volume of 50 μL of DMEM per well. The cells were incubated with 5% CO2 at 37 °C for 2 d. Then, the medium was replaced with FBS-free DMEM, and 2 μg/mL BFT-2 was added to a final volume of 1 mL. After that, morphological changes were observed by optical microscopy at 20, 40, 60, 90 and 120 min, respectively.
Grouping for the animal experiment: After 1 wk of adaptive observation, the mice were divided into four groups: the blank control group (BC), the AOM/DSS group (AD), the AOM/DSS + low-dose toxin group (ADLT), and the AOM/DSS + high-dose toxin group (ADHT). Mice in the BC group drank and ate freely, while mice in the AD group were intraperitoneally injected with AOM (0.2 mg/kg) on the first day of the first week. From the second week onwards, the mice in the AD group were given 2% DSS-free drinking water, which lasted 1 wk. At the third week, the water provided for mice in the AD group was switched to normal drinking water. In addition, a 3-wk period was considered to be one cycle and the process lasted for three cycles in total. Based on the AD group, mice in the ADLT group and the ADHT group received intra-gastric administration of 10 μg and 20 μg BFT-2 on the first day of DSS drinking, and mice in the other groups were given an equal volume of saline as the control[13,14].
Observation of the general condition of the animals: The general conditions of the mice were observed during the experiment, including hair color, mental status, activities, feeding, defecation, nutritional status, presence or absence of anal bleeding, and rectal prolapse. Body weight of the mice was weighed and recoded once a week. At the end of wk 14, the mice were sacrificed by cervical dislocation after fasting for 12 h.
Status of tumor formation: The abdominal cavity was fully exposed after the mice were sacrificed, and the large intestine was dissociated and completely removed. Along the longitudinal axis, the intestinal tract was split and flattened. Then, PBS buffer was used to wash the intestinal tract. Finally, the number, location and the size of the tumors, and the length of the large intestine were recorded.
Histopathological examination: Normal tissue of the colorectal mucosa and tumor tissues were selected and fixed in 4% paraformaldehyde solution. After fixing, the following steps were performed and included dehydration, transparency, embedding with paraffin, and HE staining. Finally, the lesion level of the induced CRC was evaluated for each group.
Immunohistochemical examination of normal colorectal mucosal tissues and tumor tissues: The expressions of Ki-67 and Caspase-3 in the specimens of normal colorectal mucosal tissues and tumor tissues were measured by SP staining. Ki-67 is expressed in the nucleus, and Caspase-3 is expressed in the cytoplasm. Semi-quantitative scoring was used. Based on the staining intensity, the specimen was classified according to the following levels: negative staining was considered as 0 points, weakly positive staining (+, pale yellow) as 1 point, moderately positive staining (++, brown) as 2 points and strongly positive staining (+++, tan) as 3 points. Furthermore, the specimen was scored based on the proportion of the number of positive cells. According to the range of positive staining, 5% was considered as 0 points, 5%-25% as 1 point, 26%-50% as 2 points, and 51%-75% as 3 points; greater than 75% was considered as 4 points. Statistics were performed for positive immunoreactivity when the product of the staining intensity and the proportion of the range of positive observations was greater than or equal to 2.