Bacterial strains, plasmids and cell lines
Competent Escherichia coli DH5α cells and plasmids pMD-18 T (TaKaRa) and pcDNA5/FRT (pDF) (Life Technologies) were used for cloning and protein expression. Flp-In-CHO cells were purchased from Life Technologies. WISH cells, HeLa cells, K562 cells and vesicular stomatitis virus (VSV) have been described previously [12]. RhIFN-α2b was obtained from Yuan-Ce Corporation (Beijing, China). Trypsin and fetal bovine serum (FBS) were purchased from Gibco.
Recombinant eukaryotic rhIFN-λ1 vector construction
The rhIFN-λ1 gene was amplified via PCR from a pMD18T-λ1 plasmid template from Invitrogen using the following primers: 5′-gctagcatggctgcagcttggaccgtggtgctggtgac-3′ (sense primer: NheI, recognition site underlined) and 5′-aagcttttatcaggtggactcagggtgggttgacgttc-3′ (antisense primer: HindIII, recognition site underlined). The reaction conditions were 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 60 s; and 72 °C for 10 min. The amplicon was then inserted into pcDNA5/FRT (pDF) using the primer restriction sites. The recombinant plasmid, named pDF-λ1, was identified by digestion with NheI and electrophoresis of the digestion products, and was confirmed via sequencing (performed by the Beijing Tsinke Biotechnology Company). The recombinant plasmid was amplified and purified using an EndoFree Plasmid Maxi Kit (Qiagen).
DNA transfection and screening for high productivity cells
Flp-In-CHO cells (Life Technologies) were maintained in F12 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) at 37 °C in a humidified incubator containing 5% CO2. The cells were transfected with 1 μg plasmid (pDF-λ1:pOG44 was 1:9) using Lipo 2000 (Life Technologies) according to the manufacturer’s protocol.
To confirm expression of the recombinant protein, 48 h after transfection, the cells were fixed with methanol, washed three times in PBS, and then incubated with an anti-IFN-λ1 mouse monoclonal antibody (diluted 1:1000; Abcam) at 37 °C for 1 h. After washing three times with PBS, the cells were incubated with a secondary rabbit monoclonal anti-mouse IgG antibody (KPL) conjugated with FITC (diluted 1:2000) at 37 °C for 1 h. Finally, the cells were washed three times in PBS and observed using fluorescence microscopy. The transfected cells were screened in growth culture medium supplemented with 500 μg/ml hygromycin B (Invitrogen). Single clones were obtained by the limited dilution method in 96-well cell culture plates. The quantity of rhIFN-λ1 was evaluated using an ELISA kit (Abcam).
Identification of clones stably expressing rhIFN-λ1
Clones were cultured in medium supplemented with 500 μg/ml hygromycin B for ten passages. Genomic DNA extracted with a QIAamp DNA Mini Kit (Qiagen) was amplified via PCR and used to determine cell stability. The rhIFN-λ1 contents of the culture media of the 5th and 10th passages were evaluated using an ELISA kit (Abcam). In addition, rhIFN-λ1 protein was detected in the cell culture media of the 5th and 10th passages via western blot. The primary antibody was a mouse anti-human IFN-λ1 antibody and the secondary antibody was goat anti-mouse IgG-HRP (Zsbio).
Purification of the rhIFN-λ1 protein
RhIFN-λ1 CHO cell culture medium was harvested via centrifugation at 15,000×g for 30 min at 4 °C. Protein was precipitated from the culture medium for 2 h at room temperature after the addition of 40–85% ammonium sulfate. The precipitate was dissolved in buffer A (50 mM phosphate buffer, pH 7.0, 5 mM EDTA) and dialyzed overnight at 4 °C.
The first step was cation exchange chromatography. An SP Sepharose Fast Flow column was equilibrated with 5–10 column volumes of buffer A before the addition of the sample at a rate of 1 ml/min. The column was washed with buffer A to elute the non-bound protein until the absorbance was near zero, which indicated no protein in the eluate. Then buffer B (buffer A containing 0 to 1 M NaCl) was used as a linear gradient to elute rhIFN-λ1 from the column. The elution peak was determined from the rising, plateau and falling absorption values.
The fractions were gathered in different tubes and concentrated using a centrifugal concentrator (Merck). The target protein in the purified samples was then identified using SDS-PAGE and western blot.
A Blue Sepharose 6 Fast Flow affinity column was equilibrated with 5–10 column volumes (column volume was 25 ml) of start buffer (buffer A containing 0.15 M NaCl). Purified sample containing rhIFN-λ1 was added at a rate of 1 ml/min. Unbound protein was washed off the column using start buffer. Then buffer A containing 2 M NaCl (elution buffer A) was used to obtain elution peak 1, and the column was eluted with buffer A containing 50% ethylene glycol to obtain elution peak 2.
Proteins from both elution peaks were condensed via ultrafiltration and assessed using SDS-PAGE and western blot. The rhIFN-λ1 solution was equilibrated with buffer A and finally molecular sieve chromatography was performed using Sephacryl S-100 gel equilibrated with buffer A. After adding the sample at a rate of 0.5 ml/min, the protein was collected in a different tube and concentrated. Because of the different sized molecules in the eluate, fractions with different absorption peaks were collected separately. The various fractions of the elution were assessed using SDS-PAGE and western blot.
Anti-viral assay for rhIFN-λ1 in WISH cells
WISH cells were seeded in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS at a density of 104 cells/well in 96-well plates and left overnight. The cells were incubated with successive 40- to 49-fold dilutions of rhIFN-λ1, commercially available rhIFN-λ1 (R&D), or 1000 U/ml rhIFN-α2b (as the standard) for 24 h before being challenged with VSV at the 100 tissue culture infective dose (TCID50) in each well.
Cytopathic effects were observed after incubation with the virus for 16–20 h. Crystal violet was added to the wells when all the control cells were dead and the absorbance at 570 nm was measured to determine the degree of cytopathy in different wells.
The anti-viral activity of rhIFN-λ1 was calculated according to the interferon standard, which was defined as one unit of activity when 50% of cells were dead. Using the same method, we detected the anti-viral activity of rhIFN-α2b, rhIFN-λ1, and rhIFN-λ1 (R&D) in A549 cells challenged with VSV.
Assay for anti-influenza virus activity of rhIFN-λ1
RhIFN-α2b and rhIFN-λ1 were successively diluted to 40- to 49-fold dilutions and incubated with A549 cells for about 16 h. Then anti-influenza virus activity was measured after infection with 100TCID50 of influenza virus (A/PR/8/34, H1N1) for 16–20 h, as described above, using crystal violet to measure the degree of cytopathy.
Determination of anti-proliferation activity of rhIFN-λ1
Anti-proliferation activity was determined using a CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega) according to the manufacturer’s instructions. HeLa cells were seeded in 96-well plates (5 × 104 cells/well) and then cultured in DMEM containing 10% FBS. After 6 h, the culture medium was discarded and a dilution series of rhIFN-λ1 or rhIFN-α2b was added. After incubation for 72 h at 37 °C, 20 μl of One Solution Reagent was added to each well and incubated for 4 h. The optical density was then measured at A490.
Determination of rhIFN-λ1 activity to promote natural killer cell cytotoxicity
Natural killer cell (NK) cytotoxicity was determined using the dehydrogenase releasing method. Normal human peripheral blood lymphocytes were adjusted to 106 cells/ml using DMEM containing 10% FBS. The NK cells were mixed with K562 target cells at a ratio of 100:1 and then treated with a dilution series of rhIFN-λ1 or rhIFN-α2b for 4 h at 37 °C. The supernatant was then obtained by centrifugation for 10 min at 3000×g. Lactate dehydrogenase content in the supernatant, which represents NK cytotoxicity activity, was measured with the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega).
