br The total RNA was extracted from cells using
The total RNA was extracted from cells, using TRIzol (Takara, Japan). RNA concentration was measured by NanoDrop 2000 spectrometer. 500 ng of total RNA from each sample was reversely transcribed to cDNA using Prime Script RT Master Mix (Takara, Japan). RT-PCR was performed in the following conditions: 1 cycle at 94 °C for 30 s, and 40 cycles at 94 °C for 5 s, 60 °C for 15 s, and 72 °C for 10 s. The mRNA level for each gene was normalized to GAPDH. Primer sequences are showed as following:
2.10. Western blotting analysis
HCT-8/Fu cells were treated with different concentration of MBP-FMBP-2 for 48 h. The total protein was extracted by lysing cells in lysis buffer for 30 min on ice, and then centrifugated for 10 min at 13,000 rpm. Equal amounts of protein (60 ng) from each sample were separated by SDS-PAGE (10%), and then the protein was transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were
incubated with primary 3X FLAG Peptide at 4 °C overnight, and then the PVDF membranes were incubated with secondary antibodies at room temperature for 2 h. Then the protein bands were visualized using en-hanced chemoluminescence.
2.11. Statistical analysis
Data analysis was carried out using SPSS 17.0 software. Data were expressed as means ± s.d. of at least three separate tests. Student's t-test was used for single variable comparisons. Comparisons of means of ≧3 groups were performed by analysis of variance (ANOVA), followed by Tukey's posthoc test. The values of b0.05 (p b 0.05) and 0.01 (p b 0.01) were considered significant and highly significant, respectively.
3.1. Clone of three truncated segments of FMBP
In order to screen anti-colon cancer active fragment of FMBP, three different gene segments were cloned and expressed by genetic engi-neering method. With the total cDNA of foxtail millet as the template, three truncated gene segments of FMBP were cloned, and respectively named FMBP-1, FMBP-2 and FMBP-3 (Fig. 1A, B). The positive clone products were digested by double restriction enzymes Bma H I and Hind III, and the plasmid (pMal-s) was digested by the same restriction enzymes. The PCR products and prepared plasmid were linked by T4 DNA ligase, and then we obtained recombinant plasmids (pMal-s-FMBP-1, pMal-s-FMBP-2 and pMal-s-FMBP-3). Next, the recombinant plasmids were respectively identified with Bma H I and Hind III, and the expected bands of 242 bp, 549 bp and 279 bp were respectively ob-served by agarose gel electrophoresis assay (Fig. 1C–E). These results in-dicate that three truncated segments of FMBP are inserted into the
Fig. 1. Cloning of three truncated segments of FMBP. (A) Abridged general view of three segments of FMBP. (B) Three amplified gene segments of FMBP. line 1: FMBP, line 2: FMBP-2, line 3:
1, line 3: Restriction enzymes analysis of recombinat pMal-s-FMBP-1. D: line 1: pMal-s, line 2: recombinat pMal-s-FMBP-2, line 3: Restriction enzymes analysis of recombinat pMal-s-
FMBP-2. E: line 1: pMal-s, line 2: recombinat pMal-s-FMBP-3, line 3: Restriction enzymes analysis of recombinat pMal-s-FMBP-3.
expression vector (pMal-s) successfully. Furthermore, the results of DNA sequences analysis of the recombinant plasmids also indicate that the reading frames are correct (data not shown).
3.2. Expression and purification of the truncated recombinant fragments
To confirm the optimal induction temperature and improve the ex-pression levels of MBP-FMBP-1, MBP-FMBP-2, and MBP-FMBP-3, the dif-ferent induction temperature of 16 °C, 30 °C and 37 °C were tested. The result showed that all proteins were expressed when incubated at 30 °C or 37 °C for 4 h, and the expression levels of the proteins were the highest under the condition of 37 °C (Fig. 2A–D), but no expression at 16 °C (data
not shown). The vector pMal-s contains malE gene of Escherichia coli encoding MBP fusion protein. According to the affinity principle of MBP to maltose, the MBP-FMBP-1, MBP-FMBP-2 and MBP-FMBP-3 were puri-fied using Amylose Resin affinity chromatography column. As shown in Fig. 2E and F, the purity of all proteins was about 90%.
To identify the anti-tumor activities of MBP-FMBP-1, MBP-FMBP-2 and MBP-FMBP-3, MTT assay was carried out. HCT-116 and DLD1 cells were treated for 48 h respectively with different concentration (0, r>