Antiviral activity of mink interferon alpha expressed in the yeast Pichia pastoris

Abstract

Many viruses can cause infections in mink, including canine distemper virus, mink enteritis virus, and Aleutian disease virus. Current treatments are ineffective, and these infections are often fatal, causing severe economic losses. As antiviral drugs may effectively prevent and control these infections, recent research has increasingly focused on antiviral interferons. Herein, the gene encoding a mature mink interferon alpha (MiIFN-α) was synthesized according to the P. pastoris preference of codon usage and a recombinant plasmid, pPICZαA-MiIFN-α, was constructed. pPICZαA-MiIFN-α was linearized and transformed into the P. pastoris X33 strain, and zeocin-resistant transformants were selected. Protein expression was induced by methanol. SDS-PAGE and western blot analyses showed that a 25-kDa fusion protein was expressed in the culture supernatant. Antiviral activity of the expressed protein was determined using cytopathic effect inhibition (CPEI). The purified MiIFN-α significantly inhibited the cytopathic effect of vesicular stomatitis virus with a green fluorescent protein (VSV-GFP) in F81 feline kidney cells, with an antiviral activity of 6.4 × 10⁷ IU/mL; it also significantly inhibited MEV replication in F81 cells. MiIFN-α antiviral activity against VSV-GFP was significantly reduced on treatment with pH 4 and pH 10 conditions for 24 h (p < 0.01). Serum MiIFN-α concentrations in rat were measured using enzyme-linked immune-sorbent assay; MiIFN-α concentrations in rat serum peaked at ~36 h after injection. A high dose of MiIFN-α was safe for use. There were no significant differences in body temperature, tissue changes, and lymphocyte, total white blood cell, and central granulocyte counts between the injected and control groups (p > 0.05). These findings lay a foundation for the large-scale production of recombinant MiIFNs.

Keywords: antiviral activity; interferon-alpha; mink; serum drug concentration; yeast expression.

Figure 1

Construction and identification of an MiIFN-α recombinant yeast expression vector. (A) Digestion of pUC57-MiIFN-α recombinant plasmid with EcoR I and Xba I. (B) Identification of pPICZα-MiIFN-α recombinant plasmid by restriction enzyme digestion with EcoR I and XbaI. (C) PCR identification of pPICZα-MiIFN-α plasmid.

Figure 2

Nucleotide alignment between the original and optimized sequences. Nucleotide homology between the optimized and original sequences reached 74.8%. Black areas indicate differences in nucleotides.

Figure 3

Identification of recombinant MiIFN-α expression and purification by SDS-PAGE and western blotting. (A,B) Yeast expression and purification of MiIFN-α was observed by SDS-PAGE. Lane 1: empty vector-induced control; Lane 2: recombinant yeast non-induced control; Lane 3: 6 h induced supernatant; Lane 4: 12 h induced supernatant; Lane 5: 24 h induced supernatant; Lane 6: 48 h induced supernatant; Lane 7: 72 h induced supernatant; Lane 8: 96 h induced supernatant; Lane 9: 108 h induction of supernatant; Lanes 10 and 11: protein molecular weight standards; Lanes 12–14: Eluted with binding buffer; Lanes 15 and 16: recombinant MiIFN-α purified by NTA-Ni chromatography at elution buffer pH 7.4. (C) Identification of the expressed product by western blotting. Lane 17: protein molecular weight standards; Lane 18: empty vector induced control; Lane 19: purified target.

Figure 4

BCA standard curve drawing. BSA dilution concentrations ranged from 0.001 to 0.01 mg/mL. The BCA standard curve indicates that the linear correlation of the protein concentration and OD_450nm value was y = 0.0242x-0.0014 (R² = 0.9926).

Figure 5

Antiviral activity of MiIFN-α against VSV-GFP in F81 cells. (A–D) Changes in the green fluorescence intensity indicating the antiviral activity of the canine IFN-α standards of 488.28 IU/mL (4⁶-fold), 122.07 IU/mL (4⁷-fold), 30.52 IU/mL (4⁷-fold), and 7.63 IU/mL (4⁷-fold). (A1) MiIFN-α 0.36 ng/mL(4⁶-fold). (B1) MiIFN-α 0.091 ng/mL(4⁷-fold). (C1,C2) MiIFN-α 0.023 ng/mL (47-fold). (A2,B2) Uninfected cells. (D1,D2) Virus controls.

Figure 6

Observation of cell staining with crystal violet. Adherent F81 cells were stained with 0.5% crystal violet, which rendered the viable cells blue-violet; the non-colored dead cells were washed away with the effluent. Results of absorbance detection at 540 nm show that the antiviral activity of standard canine IFN-α (2 × 10⁶ IU/mL) decreased significantly at 4⁶ dilutions and that of mink IFN-α slowly decreased at 4⁹ dilutions.

Figure 7

Detection of the metabolism of MiIFN-α in rats. (A) Standard curve drawing of canine IFN-α ELISA kit. (B) OD_450nm absorbance at different time points. (C) Residual concentration of interferon-α at different time points in rats.

Figure 8

MiIFN-α inhibits MEV SMPV-11 replication in vitro. (A) MEV Standard curve of quantitative real-time PCR. (B) Changes in Cq values. (C) Changes in copy number. ****, P ≤ 0.0001.

Figure 9

Effect of various pH levels on the antiviral activity of MiIFN-α. Antiviral activity of mink IFN-α in a F81/VSV system treated at 4°C for 24 h at pH 2, 4, 6, 7.4, 8, 10, 12, and for 28 h at pH 7.4; p < 0.01. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.