Characterization of bovine interferon α1: expression in yeast Pichia pastoris, biological activities, and physicochemical characteristics

Abstract

A bovine interferon α (BoIFNα) gene that included signal sequence was amplified from bovine liver genomic DNA. The gene was named BoIFN-α1 according to the position at which the encoded gene of the bovine IFN was located in the bovine genome. The sequence included a 23-amino-acid signal peptide and a 166-amino-acid mature peptide. The structural characteristics and phylogenetic relationships of the BoIFN-α1 gene were analyzed. A recombinant mature BoIFN-α1 (rBoIFN-α1) was expressed in the yeast Pichia pastoris. Physicochemical characteristics and antiviral activity were determined in vitro. Recombinant BoIFN-α1 was found to be highly sensitive to trypsin and stable at pH 2.0 or 65°C. It also exhibited antiviral activity, which was neutralized by a rabbit anti-rBoIFNα polyclonal antibody. This study revealed that rBoIFN-α1 has the typical characteristics of IFNα and can be used for both research and industrial application.

FIG. 1.

The results of secondary structure prediction. The sequence underlined with a single line is a signal sequence, the arrow denotes the signal sequence cleavage site, and underlined with wavy lines are the two highly conserved domains. Cysteine residues forming disulfide bonds are marked with gray circles. The conserved amino-acid residues Arg-33 and Tyr-123 are marked with a gray box and a white circle. Letters A to E refer to the α-helices in bovine IFN-α₁. IFN-α₁, interferon α₁.

FIG. 2.

Phylogenetic tree based on nucleotide sequences of bovine IFN-α₁ and type I IFN from different species by the neighbor-joining method. GenBank accession numbers are as follows: human IFNα1 (NM_024013), human IFNα2 (NM_000605), human IFNα6 (NM_021002), human IFNα7 (BC074992), murine IFNα1 (NM_010502), murine IFNα2 (NM_010503), murine IFNα4 (NM_010504), murine IFNα5 (NM_010505), porcine IFNα1 (DQ249000), porcine IFNα2 (DQ249002), porcine IFNα7 (DQ872660), porcine IFNα8 (DQ248999), bovine IFN-α₁ (XP_001251758.1), bovine IFNαA (EU276064), bovine IFNαB (M10953), bovine IFNαC (NM_174085), bovine IFNαF (NM_001172042), bovine IFNαG (EU276064), horse IFNβ (NM_001172040), and sheep IFNβ (JX458084). The IFNβ of horse and sheep were selected as outgroups.

FIG. 3.

Polymerase chain reaction determination of the transformants of Pichia pastoris containing target gene. (A) The transformed cells of yeast containing pPICαA-BoIFN-α₁ with the specific forward/reverse primers. (B) The transformed cells of yeast containing pPICαA-BoIFN-α₁ with the specific reverse/5′AOXI primers. BoIFN-α₁, bovine interferon α₁.

FIG. 4.

SDS-PAGE and western blotting analysis on purified rBoIFN-α₁ and rBoIFNαA. (A) Purified rBoIFN-α₁ and rBoIFNαA separated on 15% SDS-PAGE and stained with Coomassie Brilliant blue G-250. M, sizes (KD) of molecular-weight markers; Lane 1: supernatant of Pichia pastoris culture transformed with pPICZaA plasmid (control); Lane 2: supernatant of P. pastoris culture transformed with pPICαA-BoIFN-α₁ plasmid number induced; Lane 3: rBoIFN-α₁; Lane 4: rBoIFNαA. (B) Western blot analysis of rBoIFN-α₁ and rBoIFNαA with a goat anti-rabbit IgG conjugated to horseradish peroxidase antibody against rabbit anti-bovine IFNα polyclonal antibody. M, sizes (KD) of molecular-weight markers; Lane 1: supernatant of P. pastoris culture transformed with pPICZaA plasmid (control); Lane 2: rBoIFN-α₁; Lane 3: rBoIFNαA. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; rBoIFN-α₁, recombinant mature BoIFN-α₁.

FIG. 5.

Characterization of rBoIFN-α₁ biological activity. (A) Antiviral activity of rBoIFN-α₁ in MDBK/VSV system. (B) Neutralization of antiviral activity with anti-BoIFNα polyclonal antibody in MDBK/VSV system. MDBK, Madin–Darby bovine kidney; VSV, vesicular stomatitis virus.

FIG. 6.

The important physicochemical characteristics of rBoIFN-α₁. (A) The results of pH sensitivity of rBoIFN-α₁. (B) The results of temperature sensitivity of the rBoIFN-α₁. **P<0.01.