Cloning vector pMD18-T vector was purchased from TransGen Biotech (Beijing, China). A375 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). NdeI, XhoI, and T4 DNA ligases were purchased from New England Biolabs, Ltd. (Beijing, China). Isopropyl-β-d-thiogalactopyranoside (IPTG) was purchased from Merck & Co., Inc. (Darmstadt, Germany). QIA quick gel extraction and nucleotide removal kits were obtained from Qiagen China Co., Ltd. (Shanghai, China). Commercial recombinant human S100B protein and S100B antibody were from Abcam (Cambridge, MA, USA). Unless otherwise stated, other reagents and materials were performed as described in the literature (7). Animals were purchased from the Experimental Animal Room of the Institute of Health and Environmental Medicine (Tianjin, China). This study was approved by the Committee of the Institute of Health and Environmental Medicine.

The gene fragment of human S100B was constructed by overlapping polymerase chain reaction (PCR) after optimization. NdeI and XhoI restriction sites were designed in sense and antisense primers, respectively. Eight oligonucleotide primers with mutual overlaps were synthesized based on the codon preference in E. coli (Table I). The NdeI restriction site (underlined) was contained in the forward primer (5′-CGCCATATGTCTGAACTGGAAAAAGCC-3′), whereas the XhoI restriction site (underlined) was contained in the reverse primer (5′-CGGCTCGAGTCACTCATGTTCAAAGAAC-3′). E. coli DH5α cells were transformed with the constructed cloning vector pMD18-S100B plasmid. Standard PCR was used to screen the positive plasmid. To identify the integrity of S100B, pMD18-S100B plasmid was DNA sequenced (Invitrogen Life Technologies, Shanghai, China). The expression vector pET32a-S100B plasmid was constructed and then ligated with the coding region of human S100B from the pMD18-S100B plasmid.

The expression vector pET32a-S100B plasmids were transformed into E. coli BL21 (DE3). Positive E. coli was inoculated in 5 ml of Luria-Bertani (LB) media containing 50 µg/ml ampicillin. IPTG was used to induce the expression of human S100B protein. Bacterial lysate was collected to isolate the recombinant S100B. S100B was purified by ion-exchange chromatography column (HiTrap™ DEAE FF; binding buffer: Tris-HCl 0.02 mol/l, pH 8.8; and elution buffer: 500 mmol/l NaCl Tris-HCl 0.02 mol/l, pH 8.8; GE Healthcare Life Sciences, Chalfont, UK) in accordance with the manufacturer's instructions, desalted, and freeze dried. Protein concentration was determined by bicinchoninic acid (BCA) method (BCA Protein Assay kit; Tiangen Biotech Co. Ltd., Beijing, China) (7), and S100B protein was characterized by western blot analysis.

Cell motility and invasion assays were performed in a Transwell chamber to detect whether the recombinant S100B protein can stimulate cell migration and invasion (8). In accordance with the manufacturer's instructions, Transwell chambers (EMD Millipore) were coated with Matrigel^®, and assays were performed. HeLa cells (1×10⁵) without S100B protein were utilized as the control group (C), whereas HeLa cells (1×10⁵) with 50 µg/ml S100B protein were used as the experimental group (E). HeLa cells (100 µl) of C or E were added to the top chambers of 24-well Transwell plates. After 12 h of incubation at 37°C, the HeLa cells at the bottom of each chamber were fixed with 0.1% v/v glutaraldehyde for 30 min, rinsed with PBS, and then stained with 0.2% v/v crystal violet for 20 min, whereas the motile cells at the top of each chamber were removed with cotton swabs. The number of migrating cells or invasive cells was calculated under ×200 magnification (Olympus CKX31; Olympus, Tokyo, Japan). The number of average cells per chamber was also determined. Duplicate experiment was performed with each assay and repeated at least thrice. Data were measured as the migration/invasion rates relative to the parental control cells.

The immunization procedure was performed using a previously described technique with slight modification (7,9,10). Female BALB/c mice (weight, 18–25 g; age, 8–10 weeks old) were provided by the Experimental Animal Room of the Tianjin Institute of Health and Environmental Medicine. Four mice were bred per cage at 22–25°C, ad libitum with tap water and standard diet, under an alternating 12 h light/dark cycle. The purified recombinant S100B (50 µg) was mixed with an equal volume of Freund's complete adjuvant, which was multipoint-injected subcutaneously on the back of the mice. Animals were immunized thrice over the course of 45 days with purified recombinant S100B protein. On the 10th day after the last injection, the mice were sacrificed by cervical dislocation, and the splenocytes were collected and fused with SP2/0 myeloma cells by using the PEG (PEG, polyethylene) method. Hybridoma cells were selected in the hypoxanthine, aminopterin, and thymidine media (purchased from Gibco Life Technologies, Grand Island, NY, USA). Positive hybridoma cell lines were obtained following three subcloning cycles and were confirmed by indirect ELISA. Hybridoma cells were injected intraperitoneally into liquid paraffin-primed female BALB/c mice (8–10 weeks old) at ~1×10⁶ cells/mouse to produce anti-S100B mAbs. Purification of antibodies was performed using HiTrap protein G HP (1 ml) (GE Healthcare Life Sciences) in accordance with the manufacturer's instructions. The binding buffer was 20 mmol/l Na₃PO₄ at pH 7.0, and the elution buffer was 0.1 mol/l Gly-HCl at pH 2.7. The purity of the antibody was determined by SDS-PAGE and western blot analysis. The affinity constants of antibodies were identified by SPR (11,12).

Antibody titers were measured by indirect ELISA (11,12). The microtiter plates were coated with ~100 µl of recombinant S100B (4 µg/ml) and then incubated at 4°C overnight. The plates were washed thrice with PBS containing 0.05% Tween-20 (PBST) and blocked with 3% BSA in PBS containing Tween-20 for 1 h at 37°C. The plates were placed in different serum dilutions from immunized mice, ascites, or cell culture supernatants for 2 h at 37°C and then incubated with HRP-coupled goat anti-mouse IgG for 1 h. The substrate used was tetramethylbenzidine (TMB). The absorbance was determined at 450 and 630 nm wavelengths, respectively. The specification of the mAbs were examined using competitive ELISA (11,12) (Table II).

The SDS-PAGE procedure was performed as previously described with slight modification (13). The concentrations of the resolving and stacking gels were 15 and 5%, respectively. The purified protein was transferred to a nitrocellulose membrane with a semi-dry electrophoretic transfer device (Jim-X Biotechnology Co., Ltd., Dalian, China). The membrane was blocked with 0.5 ml of 10% BSA in 4.5 ml of Tris-buffered saline/Tween-20 buffer (10 ml of 1 mol/l Tris+HCl at pH 7.5, 8.8 g of NaCl, 1000 ml of ultra-pure water, and 1 ml of 20% Tween-20) and incubated with rabbit polyclonal antibody against human S100B for 2 h at 25°C. Following the HRP-coupled goat anti-rabbit IgG, a secondary antibody was added and incubated for 45 min.

To identify the cross reactivity (CR) of the recombinant S100B and its analogs, indirect ELISA was utilized as previously described (12,13). As coated antigens, recombinant S100B, recombinant rat S100A4, human S100A4, and S100A1 were all dissolved in PBS. A diluted antibody protein of S100B was incubated with these coated antigens for 2 h at 25°C. Afterward, HRP-conjugated goat anti-mouse polyclonal antibody was added and then incubated for 45 min at 25°C. Finally, the absorbance was determined at 450 nm wavelength by using a microplate reader (Multiskan MK3; Thermo Fisher Scientific, Waltham, MA, USA).

The mAb affinity against S100B was estimated by surface plasmon resonance (SPR, AutoLab ESPRIT, Utrecht, The Netherlands). In accordance with the manufacturer's instructions, S100B was immobilized on a carboxylated sensor chip (Metrohm Auto Laboratory). Extracted mAbs were added to the immobilized chip surface. Data were analyzed using Kinetic Evaluation 5.0 software (Metrohm Auto Laboratory) and Autolab ESPRIT Data Acquisition 4.3.

Cell proliferation assay was determined using Cell Counting Kit-8 (CCK-8; Dojinoo Techno Research Park, Mashiki-machi, Japan). A375 cells (5×10⁴) of C or E were added to the 96-well plates and incubated for 24 h at 37°C. Different concentrations (100 µg/ml, 10 µg/ml, 1 µg/ml, 100 ng/ml, 10 ng/ml, and 1 ng/ml) of S100B mAbs (100 µL) were added to the plates as the experimental group (E), whereas DMEM medium (100 µl) was used as the control group (C). The absorbance was determined at 450 nm wavelength by using a microplate reader (Multiskan MK3; Thermo Fisher Scientific).

Cell apoptosis assay was performed using a Histone ELISA kit (from Biosoure™; Invitrogen, Carlsbad, CA, USA). A375 cells (1×10⁵) were added to the 96-well plates. Different concentrations (1 mg/ml, 100 µg/ml, 10 µg/ml, 1 µg/ml, 100 ng/ml, 10 ng/ml, and 1 ng/ml) of S100B mAbs (100 µl) were added to the plates as the experimental group (E), whereas DMEM medium (100 µl) was used as the control group (C). The absorbance of the lysates of the cells was determined at 405/490 nm wavelength in accordance with the manufacturer's instructions.

To evaluate the effect of S100B mAbs on the expression of S100B and p53 in A375 cells, western blot analysis was performed. Anti-human S100B mAbs prepared in the present study was added to A375 cells in a culture flask (25 cm). The concentrations of S100B mAbs in the medium are 10 µg/ml and 100 ng/ml as the experimental group (E), whereas DMEM medium was used as the control group (C). After incubation at 37°C for 24 h, A375 cells were collected and disrupted for 30 min. The entire set of proteins was preserved at −70°C. Anti-human S100B mAbs (prepared and purchased separately) were used as first antibodies in western blot analysis.

Data are shown as means ± SD. All results were analyzed using SPSS version 17 (SPSS, Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The human S100B protein natural gene sequence (Genbank accession no. NM 006272.2) was optimized using the E. coli codon. Sixteen rare codons of the S100B original gene were superseded with synonymous high-frequency codons in an optimized gene that encodes S100B (Fig. 1).

The size of the PCR products was 250–300 bp, which conforms to the expected size of 279 bp (Fig. 2A). By Ndel and Xhol digestion, the S100B gene was successfully ligated into the pMD18-T vector. Agarose gel analysis of the digestion of pMD18-S100B vector is indicated in Fig. 2B. Through DNA sequencing, the synthesized S100B gene was consistent with the optimized S100B gene without point mutation and frameshift mutation (Fig. 2D).

S100B gene was synthesized, and the recombinant expression plasmid pET32a-S100B was constructed successfully (Fig. 2C). The results of the restriction enzyme digestion showed that the S100B gene plasmid contained the full coding sequence, and the open reading frame was correct. The recombinant plasmid pET32a-S100B was transformed into expression strain E. coli BL21 (DE3) and was induced by IPTG. The molecular weight of the S100B fusion protein was 11.5 kDa. The expressed recombinant S100B was identified by SDS-PAGE and western blotting using a rabbit anti-human S100B antibody. The recombinant S100B protein was successfully expressed in E. coli BL21 (DE3) because of the reaction with the antibody (Fig. 2F and G) and was effectively purified by ion-exchange chromatography (Fig. 2H).

The function of human recombinant S100B was detected by Transwell chamber test. The results showed that the recombinant S100B protein increased the invasion of HeLa cells 2.8 times (Fig. 3A-C) and the migration 3.5 times (Fig. 3D-F), indicating that S100B protein can promote HeLa cell invasion and migration. The purified S100B proteins demonstrate a potential biological activity and can improve the ability of tumor cell invasion and migration.

Murine mAbs against S100B were prepared to further explore the function of human S100B. Three hybridoma cell lines, namely, 4E₁₀F₁₁C₁₁, 3D₂E₅F₇, and 4F₃A₅D₅, were subsequently formed from the immunization of mice with soluble S100B as the antigen. Highly concentrated S100B mAbs were prepared from BALB/c mouse ascites and purified by protein G affinity chromatography. These cell lines stably produced anti-S100B mAbs (Table II). The specificity of the S100B mAbs was determined by western blot analysis (Fig. 2E).

CR is a key parameter used to assess the specificity of an antibody because the human S100B exhibits similar structures and functions of the following proteins: Human S100A4, Mouse S100A4, Human S100A1, and Human S100A1. The data summarized in Table III indicate that this antibody achieved little CRs to mouse S100A4, human S100A1, and S100B.

To determine the association rate constant, 3D₂E₅F₇ mAb was properly diluted in PBS and analyzed by SPR at different concentrations (Fig. 4). The equilibrium dissociation constant (KD) for the 3D₂E₅F₇ clone was determined independently by Kinetic Evaluation 5.0 software. The KD of 3D₂E₅F₇ was ~4.72×10⁻⁸ mol/l.

We examined the effect of S100B mAbs on cell proliferation of A375 cells. The results showed that cell proliferation of A375 cells was inhibited by anti-human S100B mAbs to some extent. Different concentrations of mAbs exert various effects on cell proliferation. The effect of mAbs on cell proliferation of A375 cells increased from low to high concentration of anti-human S100B mAbs (Fig. 5A).

To demonstrate the effect of anti-human S100B mAbs on cell apoptosis of A375 cells, cell apoptosis assay was examined in accordance with the manufacturer's instructions in the ELISA kit. The results in Fig. 5B suggest that with increasing anti-human S100B mAb concentration added to the cell culture medium, the amount of apoptosis in A375 cells was significantly increases compared with that of the control group.

The results indicated that with increasing anti-human S100B mAb concentration added to the cell culture medium, the expression of S100B in A375 cells decreased, whereas the expression of p53 in A375 cells increased significantly (P<0.05). In this study, the use of commercially purchased S100B mAb was compared with that of self-prepared S100B mAb. The results presented a similar trend. The above data illustrated that anti-human S100B mAb plays an important role in decreasing the expression of S100B and increasing the expression of P53 protein in A375 (Fig. 6).