Identification and characterization of a rabbit novel IFN-α unlocated in genome

Gao, Mingchun; Liu, Ying; Guo, Yongli; Wang, Yujiao; Dai, Haiyue; Song, Zhifeng; Wang, Junwei; Wang, Han

doi:10.1016/j.dci.2017.09.016

Cited by 5 publications

(4 citation statements)

References 37 publications

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“…The wells without viruses were considered mock–treated controls, and the wells without IFNs were designated as virus controls. One unit of interferon activity was defined as the amount required to inhibit the destruction of the cell monolayer by 50% [ 15 ]. Rabbit anti–bovine and rabbit anti–human IFN–λ3 PAb were diluted in a gradient and mixed with 100 U/mg/boIFN–λ3.…”

Section: Methodsmentioning

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%

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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: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Zhang

Guo

et al. 2023

Viruses

Self Cite

View full text Add to dashboard Cite

show abstract

“…The wells without viruses were considered as the mock-treated controls, and the wells without IFNs were designated as the virus controls. One unit of interferon activity was defined as the amount to inhibit the destruction of the cell monolayer by 50% [14].Rabbit anti-bovine and Rabbit anti-human IFN-λ3 PAb were diluted gradiently and mixed with 100 U/mg/ boIFN-λ3. Each PAb was diluted in a 96-well plate, and the inhibition effect of PAb on IFN was determined at 37 °C for 1 h. Rabbit anti-bovine IFN-α PAb was used as a negative control [15].…”

Section: Antiviral Activity Assaymentioning

confidence: 99%

“…The boIFN-λ3/λ3 V18M samples adjusted to pH2.0, 4.0 and 10.0, 12.0 with HCl and NaOH were incubated at 4 °C for 24 h, then the pHs were 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. MDBK/VSV system was used to assess the antiviral activity with respect to that of the untreated boIFN-λ3/λ3 V18M samples [8,14,16].…”

Section: Physicochemical Characteristics Analysis Of Boifn-λ3/λ3 V18mmentioning

confidence: 99%

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

An¹,

Zhang²,

Guo³

et al. 2023

Preprint

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Type III interferon (IFN-λs) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragements of bovine ifn-λ (boifn-λ) gene was synthetized after codon optimization. The boifn-λ gene was then amplified by splicing by overlap extension PCR (SOE PCR), resulting in serendipitous acquisition of the mutated boIFN-λ3V18M. Recombinant plasmids pPICZαA-boIFN-λ3/λ3V18M were constructed and the corresponding proteins were expressed in Pichia pastoris with high-level extracellular soluble form. Dominant expression strains of boIFN-λ3/λ3V18M were selected by Western blot and ELISA, cultured on a large scale, and the recombinant proteins purified by ammonium sulphate precipitation and ion exchange chromatography, yielding 1.5g/L and 0.3 g/L, with 85% and 92% purity, respectively. The antiviral activities of boIFN-λ3/λ3V18M exceeded 106 U/mg, 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. Development of boIFN-λ3 and comparative evaluation with the mutant provide theoretical insights into antiviral mechanisms of boIFN-λs and provide material for therapeutic development.

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Evolutional conservation of molecular structure and antiviral function of a type I interferon, IFN-kappa, in poultry

Gao

Guo

et al. 2018

Developmental & Comparative Immunology

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Identification and characterization of a rabbit novel IFN-α unlocated in genome

Cited by 5 publications

References 37 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

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

Evolutional conservation of molecular structure and antiviral function of a type I interferon, IFN-kappa, in poultry

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