Extracellular expression and antiviral activity of a bovine interferon-alpha through codon optimization in Pichia pastoris

摘要

Interferons (IFNs) are the primary line of defense against infectious agents. In particular, IFN-alpha is an important antiviral cytokine and has a wide range of immune-modulating functions. Porcine and human IFN-a have been successfully prepared and play important roles in the prevention and therapy of viral diseases. To date, there has been limited applied research on bovine IFN-alpha. To achieve high-level expression of recombinant bovine IFN-alpha (bIFN-alpha) in Pichia pastoris for large-scale application, the bIFN-alpha gene was optimized and synthesized on the basis of codon bias of P. pastoris. Optimized bIFN-alpha (opti-bIFN-alpha) was successfully expressed in P. pastoris and directly secreted into the culture supernatant. The amount of extracellular soluble opti-bIFN-alpha was observed to be 200 mu g/mL in a shake flask. Expression efficiency of opti-bIFN-alpha was found to be about three times that of wild-type bIFN-alpha when the expression yield was compared at the same copies of the targeted gene. In addition, both the original cultural supernatant and purified opti-bIFN-alpha showed strong antiviral activity in MDBK cells (2 x 10(6) AU/mL and 1 x 10(7) AU/mg, respectively) and IBRS-2 cells (3 x 10(5) AU/mL and 1.5 x 10(6) AU/mg, respectively) against a recombinant vesicular stomatitis virus expressing the green fluorescence protein. In this study, we demonstrated high-level extracellular expression of opti-bIFN-alpha by P. pastoris. To the best of our knowledge, the opti-bIFN-alpha yield observed in this study is the highest to be reported to date. Our results demonstrated that the extra cellular opti-bIFN-alpha with strong antiviral activity could be easily prepared and purified at a low cost and that it may be a potential biological therapeutic drug against bovine viral infections. (C) 2016 Elsevier GmbH. All rights reserved.