Design, biological activity and signaling pathway of bovine consensus omega interferon expressed in Pichia pastoris

Gao, Mingchun; Guo, Yongli; Luo, Xiuxin; Du, Jiwen; Wang, Yujiao; Cao, Chen; Jun-wei, Wang; Wang, Han

doi:10.1016/j.molimm.2018.12.004

Cited by 5 publications

(6 citation statements)

References 30 publications

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“…Recombinant boIFN–λ3 is neutralized by specific antibodies, giving it the following physicochemical properties: heat resistant and acid and alkali resistant [ 9 ]. These properties are comparable to those of known type I interferons, confirming the functional similarity between IFN–λ and IFN–α [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

“…The boIFN–λ3/λ3 V18M samples adjusted to pH2.0, 4.0, 10.0, and 12.0 with HCl and NaOH were incubated at 4 °C for 24 h, and then the pH was adjusted back to the original pH of boIFN–λ3/λ3 V18M . The boIFN–λ3/λ3 V18M samples were separately incubated at 42 °C, 56 °C, and 63 °C for 2 h. The MDBK/VSV system was used to assess the antiviral activity with respect to that of the untreated boIFN–λ3/λ3 V18M samples [ 9 , 15 , 17 ].…”

Section: Methodsmentioning

confidence: 99%

“…The antiproliferative activity of boIFN–λ3 on MDBK cells was measured using the MTT assay [ 9 ]. Cells seeded in 96–well plates were treated with 0 U/mL, 10 U/mL, 100 U/mL, 1000 U/mL, and 10,000 U/mL boIFN–λ separately and then incubated with MTT for 4 h. DMSO was added after removing the culture medium, and OD490 was measured.…”

Section: Methodsmentioning

confidence: 99%

“…Pichia pastoris combines the advantages of both prokaryotic and eukaryotic expression systems, with easy preparation and purification, is suitable for high–level expression, and is characterized not only by intracellular and secretory expression but also by post–translational modifications [ 8 , 9 ]. More than 500 proteins have been successfully expressed in the P. pastoris expression system [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

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

Zhang

Guo

et al. 2023

Viruses

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Type III interferons (IFN–λs) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragments of the bovine ifn–λ (boifn–λ) gene were synthetized after codon optimization. The boifn–λ gene was then amplified by splicing using overlap extension PCR (SOE PCR), resulting in the serendipitous acquisition of the mutated boIFN–λ3V18M. The recombinant plasmid pPICZαA–boIFN–λ3/λ3V18M was constructed, and the corresponding proteins were expressed in Pichia pastoris with a high–level extracellular soluble form. Dominant expression strains of boIFN–λ3/λ3V18M 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–λ3/λ3V18M exceeded 106 U/mg, and they were neutralized with IFN–λ3 polyclonal antibodies, were susceptible to trypsin, and retained stability within defined pH and temperature ranges. Furthermore, boIFN–λ3/λ3V18M exerted antiproliferative effects on MDBK cells without cytotoxicity at 104 U/mL. Overall, boIFN–λ3 and boIFN–λ3V18M did not differ substantially in biological activity, except for reduced glycosylation of the latter. The development of boIFN–λ3 and comparative evaluation with the mutant provide theoretical insights into the antiviral mechanisms of boIFN–λs and provide material for therapeutic development.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Zhang

Guo

et al. 2023

Viruses

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“…To further explore the antiviral effect of the feIFN-ω’, the mRNA levels and protein levels of the antiviral proteins Mx1, ISG15, ISG56, and IFIT3 in the blood of the cats from each group were determined, and from these results, we clearly see that the feIFN-ω’ treatment can significantly promote the expression of antiviral proteins Mx1, ISG15, ISG56, and IFIT3, which would further exert their antiviral functions to inhibit the replication of FPV. Many studies have also demonstrated that interferon ω could stimulate the expression of the antiviral proteins ISGs [ 40 , 41 ]. In addition, we also found that the mRNA transcript levels of pro-inflammatory cytokines IL-1β, TNF-α, IL-6, and IL-12, and anti-inflammatory cytokines IL-4, and IL-10 tended to decrease ( p < 0.05 or p < 0.01) in the cats from the feIFN-ω’ treatment group, compared with the FPV infection group.…”

Section: Discussionmentioning

confidence: 99%

Cloning and codon optimization of a novel feline interferon omega gene for production by Pichia pastoris and its antiviral efficacy in polyethylene glycol-modified form

Wang

Jiang

et al. 2022

Virulence

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Feline viral diseases, such as feline panleukopenia, feline infectious peritonitis, and feline coronaviral enteritis, seriously endanger the health of cats, and restrict the development of pet industry. Meanwhile, there is a current lack of effective vaccines to protect against feline viral diseases. Thus, effective therapeutic agents are highly desirable. Interferons (IFNs) are important mediators of the antiviral host defense in animals, particularly type I IFNs. In this study, a novel feline IFN omega (feIFN-ω) gene was extracted from the cat stimulated with feline parvovirus (FPV) combined with poly(I:C), and following codon optimization encoding the feIFN-ω, the desired gene (feIFN-ω’) fragment was inserted into plasmid pPICZαA, and transformed into Pichia pastoris GS115, generating a recombinant P. pastoris GS115 strain expressing the feIFN-ω’. After induction, we found that the expression level of the feIFN-ω’ was two times more than that of feIFN-ω ( p < 0.01). Subsequently, the feIFN-ω’ was purified and modified with polyethylene glycol, and its antiviral efficacy was evaluated in vitro and in vivo , using vesicular stomatitis virus (VSV) and FPV as model virus. Our results clearly demonstrated that the feIFN-ω’ had significant antiviral activities on both homologous and heterologous animal cells in vitro . Importantly, the feIFN-ω’ can effectively promote the expression of antiviral proteins IFIT3, ISG15, Mx1, and ISG56, and further enhance host defense to eliminate FPV infection in vivo , suggesting a potential candidate for the development of therapeutic agent against feline viral diseases.

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Molecular cloning, prokaryotic expression and its application potential evaluation of interferon (IFN)-ω of forest musk deer

Yang

Cheng³

et al. 2023

Sci Rep

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Forest musk deer (Moschus berezovskii) are currently a threatened species under conservation, and the development of captive populations is restricted by health problems. To evaluate the application potential of interferon (IFN)-ω in the prevention and control of forest musk deer disease, 5 forest musk deer IFN-ω (fmdIFNω) gene sequences were successfully obtained by homologous cloning method for the first time. FmdIFNω5 was selected and recombinant fmdIFNω protein (rIFNω) was successfully expressed by pGEX-6P-1 plasmid and E. coli expression system. The obtained protein was used to stimulate forest musk deer lung fibroblasts cells FMD-C1 to determine its regulatory effect on interferon-stimulated genes (ISGs). In addition, an indirect ELISA method based on anti-rIFNω serum was established to detect endogenous IFN-ω levels in 8 forest musk deer. The results showed that there were 18 amino acid differences among the 5 fmdIFNω subtypes, all of which had the basic structure to exert the activity of type I IFN and were close to Cervus elaphus IFN-ω in the phylogenetic tree. The protein expressed was 48 kDa, and the transcription levels of all ISGs were increased in FMD-C1 cells stimulated by rIFNω, and the amount of transcription accumulation was time-dependent. Meanwhile, Anti-rIFNω serum of mice could react with both rIFNω and forest musk deer serum, and the OD450nm value of forest musk deer serum with the most obvious symptoms was the highest, suggesting that the level of natural IFN-ω in different forest musk deer could be monitored by the rIFNω-based ELISA method. These results indicate that fmdIFNω has the potential as an antiviral drug and an early indication of innate immunity, which is of great significance for the prevention and control of forest musk deer diseases.

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Design, biological activity and signaling pathway of bovine consensus omega interferon expressed in Pichia pastoris

Cited by 5 publications

References 30 publications

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

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

Cloning and codon optimization of a novel feline interferon omega gene for production by Pichia pastoris and its antiviral efficacy in polyethylene glycol-modified form

Molecular cloning, prokaryotic expression and its application potential evaluation of interferon (IFN)-ω of forest musk deer

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