Monoclonal Antibody to Bone Marrow Stromal Cell Antigen 2 Protein of Swine

Cells, antibodies, and animals

SP2/0 myeloma cells and African green monkey kidney cells (Vero E6) were obtained from Shanghai Cell Biology Institutes, Chinese Academy of Sciences (Shanghai, China). Dulbecco modified Eagle's medium, Opti-MEM cell culture medium, and fetal bovine serum were purchased from Gibco (Grand Island, NY) at 37°C in a 5% CO₂ atmosphere. Anti-FLAG antibody, anti-β-actin antibody, Alexa Fluor 488 donkey anti-mouse IgG (H + L) antibody, horseradish peroxidase (HRP)-conjugated goat anti-mouse, and p3×FLAG-CMV-7.1 plasmids were purchased from Sigma (Shanghai, China). The 6-week-old female BALB/c mice were purchased from the Shanghai Slack Laboratory Animal (Shanghai, China).

Expression and purification of rpBST-2 protein

According to the BST-2 nucleotide sequence of Sus scrofa (GenBank no. AK346678), a pair of primers (forward primer 5′-TACCCTCGAGGGATCCATGAA GGCCAACAGCAA GGGCT-3′ and reverse primer 5′-GCTTGAATTCGGATCC TCAGCTCGAAGAGCTGGAGCAGC-3′) were designed and used to amplify porcine BST-2 gene. The 336 bp fragment of BST-2 gene from small intestine tissue of piglet was amplified and inserted into the prokaryotic expression vector pCold-I. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3). The rpBST-2 protein was induced by 0.5 mM of isopropyl-β-D-thiogalactoside (IPTG) for 12 hours at 15°C and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. After centrifugation and ultrasonication, the recombinant protein was purified using Nickel Magnetic Beads (Biotool, Shanghai, China).

Immunization

Purified rpBST-2 protein (50 mg/mouse) was emulsified with an equal amount of Freund's complete adjuvant and subcutaneously injected into BALB/c mice. This was followed by three additional injections at 2-week intervals for each mouse with the same dose of the antigen plus Freund's incomplete adjuvant. Three days after the last immunization, spleen cells of immunized mice were isolated and fused with mouse SP2/0 myeloma cells.

Preparation of anti-rpBST-2 protein-specific monoclonal antibody

The anti-rpBST-2 protein serum was obtained from the immunized mice and the antibody titers were determined using indirect ELISA. Spleen cells from the immunized mice with the highest anti-pBST-2 serum antibody titer were fused with SP2/0 myeloma cells under the action of 50% PEG as fusion agents. The hybridoma cells were cultured in 96-well plates in HAT screening culture medium at 37°C in a humidified 5% CO₂ incubator. Indirect ELISA was used to filter the positive hybridomas when the cells had covered one third of the bottoms of 96-well plates. After cloning four times by limiting dilution, the positive hybridoma cells were injected into pristane-treated BALB/c mice to gain abundant ascetic fluid.

Indirect enzyme-linked immunosorbent assay

Indirect ELISA was used to determine the screen of positive hybridoma cells and antibody titer. The ELISA plates were plated with 100 mL purified rpBST-2 protein (200 ng/well) in carbonate bicarbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃ [pH 9.6]) and coated at 4°C overnight. The plates were then blocked with 5% skimmed milk in phosphate buffer with 0.05% Tween-20 (PBST) at 37°C for 1 hour. After washing thrice, the plates were incubated with 100 μL diluted cell culture supernatant or antibodies at 37°C for 1 hour. The plates were incubated with HRP-conjugated goat anti-mouse IgG with 1:10,000 dilution in PBST at 37°C for 1 hour after washing thrice in PBST. Then, the plates were incubated with 50 μL/well of TMB liquid (Amresco, Solon, OH) for 15 minutes at room temperature with protection from light. The results were read with an OD450 value after being stopped by 2 M H₂SO₄. The wells coated with purified His-tag protein served as negative control for screening the positive hybridoma cells.

Eukaryotic expression vector construction and transfection

According to the full-length BST-2 nucleotide sequence of Sus scrofa (GenBank no. AK346678), a pair of primers (forward primer 5′-CGACTCTAGAGGATCCATGTCAC CTAGTTTGTATTCCTACAGC-3′ and reverse primer 5′-ATGCCACCCGGGATCCTCAGGTCAGCAGGGCATTG-3′) were designed and used to amplify porcine BST-2 gene. The 534 bp fragment of BST-2 gene from small intestine tissue of piglet was amplified and inserted into the eukaryotic expression vector p3×FLAG-CMV-7.1 plasmids to produce the pFLAG-pBST-2 eukaryotic expression vector.

For transfection, Vero cells were seeded in six-well plates and transfected at 80% confluency with pFLAG-pBST-2 plasmids DNA in Gibco Opti-MEM cell culture medium by FuGENE HD Transfection Reagent (Promega, Madison, WI), according to the manufacturer's instructions. Empty vector transfection served as control. After 24 hours transfection, the cells were harvested and detected by Western blot analysis.

Western blot analysis

Specificity of the MAbs was confirmed by Western blot analysis. The cells' protein was collected and separated by 10% SDS-PAGE, then transferred to the nitrocellulose (NC) membrane. The membrane was blocked with 5% skimmed milk in TBST (TBS with 0.1% Polysorbate-20) for 1 hour at room temperature. The anti-FLAG antibody, anti-β-actin antibody, or anti-BST-2 MAbs were added on the NC membrane for 1 hour at room temperature. After washing thrice with TBST, the membrane was incubated with the HRP-conjugated goat anti-mouse IgG (1:6000 dilution in TBST) for 1 hour at room temperature. After washing, the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) was used for color development.

Indirect immunofluorescence assays

Vero cells were plated in a six-well plate and fixed by paraformaldehyde for 30 minutes at room temperature at 80% cell confluency. After washing twice in phosphate-buffered saline (PBS), cells were permeabilized by incubation with 0.25% Triton-100 in PBS for 5 minutes. The cells were blocked with 10% bovine serum albumin in PBS for 1 hour at 37°C after washing twice in PBS. The cells were then incubated with the anti-BST-2 monoclonal antibody (MAb) in PBS at 37°C for 1 hour. This was followed by washing four times in PBS; the cells were then incubated with Alexa Fluor 488 donkey anti-mouse IgG (H + L) antibody at 37°C for 1 hour. The cells were stained with DAPI for 5 minutes at 37°C, followed by washing twice. The cells were washed four times in PBS and then observed under fluorescence microscope.

Expression of rpBST-2 protein

The total RNA was extracted from small intestine tissue of piglet and was used for cDNA synthesis. The 336 bp fragment and the full length of porcine BST-2 gene were amplified (Fig. 1) and inserted into the expression vector. Sequencing results show that the BST-2 gene inserted into the correct position. The expression vector pCold-I containing 336 bp fragment of porcine BST-2 gene was induced by IPTG, and the high-level expression of soluble from rpBST-2 was achieved. The recombinant protein (∼13 kDa) was easily purified by His-binding resin and confirmed by SDS-PAGE and Western blot analysis. The empty vector transformed bacteria served as control (Fig. 2).

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FIG. 1.

Amplification of porcine BST-2 gene. Extracellular domain (A) and full CDS of porcine BST-2 (B) were amplificated by RT-PCR from small intestine tissue of piglet cDNA. M, DNA marker DL 2000; lane 1, product of polymerase chain reaction. BST-2, bone marrow stromal cell antigen 2; RT-PCR, reverse transcription-polymerase chain reaction.

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FIG. 2.

Expression and purification of recombinant porcine BST-2 protein analyzed by SDS-PAGE. Lane 1, supernatant proteins; lane 2, precipitation proteins; lane 3, purified rpBST-2 protein; M, PageRuler prestained protein ladder. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Generation of porcine BST-2 MAbs

The purified rpBST-2 protein was used to immunize BALB/c mice to prepare MAbs. The antisera titers were determined by indirect ELISA after four immunizations. The mouse with the highest antibody titers against rBST-2 was used for cell fusion. After subcloning four times by limiting dilution and screening, one positive MAb against porcine BST-2 protein was identified and named 1B2.

Reactivity of MAb 1B2

The MAb 1B2 cell clone was used to prepare the ascites containing MAbs. The ascites were purified using NAb™ Protein G Spin Columns (Thermo Fisher Scientific, Rockford, IL). Western blot analysis (Fig. 3) and Immunofluorescence assay (IFA) (Fig. 4) were used to identify the specificity of the MAbs against overexpressed porcine BST-2 protein in Vero cells. The results suggested that the MAb could be specifically reacted with porcine BST-2 protein.

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FIG. 3.

Western blot analyzed the specificity of prepared MAbs against porcine BST-2. Expression of overexpressed porcine BST-2 in Vero cells was detected by anti-FLAG antibody, BST-2 MAb 1B2, and anti-β-actin antibody. The protein of β-actin was detected as a control. All the experiments were repeated thrice and representative results were shown. MAb, monoclonal antibody; MAbs, monoclonal antibodies.

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FIG. 4.

Indirect immunofluorescence assays analyzed the specificity of prepared MAbs against porcine BST-2. Vero cells were plated and transfected in six-well plates. Twenty-four hours later, cells were incubated with MAb 1B2 or normal mouse antibody (negative control), followed by Alexa Fluor 488 donkey anti-mouse IgG (H + L) antibody.