Biological Activity of Optimized Codon Bovine Type III Interferon Expressed in Pichia pastoris

作者：An, Ran; Zhang, Runxiang; Guo, Yongli; Geng, Jinfeng; Si, Minglu; Wang, Shuangfeng; Gao, Mingchun^*; Wang, Junwei^*

来源：Viruses-Basel, 2023, 15(5): 1101.

DOI：10.3390/v15051101

• bovine interferon-lambda 3
• Pichia pastoris
• codon optimization
• glycosylation
• antiviral activity
• antiproliferative activity

Type III interferons (IFN-?s) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragments of the bovine ifn-? (boifn-?) gene were synthetized after codon optimization. The boifn-? gene was then amplified by splicing using overlap extension PCR (SOE PCR), resulting in the serendipitous acquisition of the mutated boIFN-?3(V18M). The recombinant plasmid pPICZaA-boIFN-?3/?3(V18M) was constructed, and the corresponding proteins were expressed in Pichia pastoris with a high-level extracellular soluble form. Dominant expression strains of boIFN-?3/?3(V18M) were selected by Western blot and ELISA and cultured on a large scale, and the recombinant proteins purified by ammonium sulfate precipitation and ion exchange chromatography yielded 1.5g/L and 0.3 g/L, with 85% and 92% purity, respectively. The antiviral activity of boIFN-?3/?3(V18M) exceeded 10(6) U/mg, and they were neutralized with IFN-?3 polyclonal antibodies, were susceptible to trypsin, and retained stability within defined pH and temperature ranges. Furthermore, boIFN-?3/?3(V18M) exerted antiproliferative effects on MDBK cells without cytotoxicity at 10(4) U/mL. Overall, boIFN-?3 and boIFN-?3(V18M) did not differ substantially in biological activity, except for reduced glycosylation of the latter. The development of boIFN-?3 and comparative evaluation with the mutant provide theoretical insights into the antiviral mechanisms of boIFN-?s and provide material for therapeutic development.

• 单位
  东北农业大学; 哈尔滨医科大学