PUBLICATION
Cloning, Exogenous Expression and Function Analysis of Interferon-γ from Gadus macrocephalus
- Authors
- Jiang, J., Gu, J., Zhan, A., Mao, M., Liu, Y., Wang, H., Mao, Y.
- ID
- ZDB-PUB-221028-32
- Date
- 2022
- Source
- Viruses 14(10): (Journal)
- Registered Authors
- Keywords
- Gadus macrocephalus, interferon γ, prokaryotic expression, yeast expression
- MeSH Terms
-
- Amino Acids/genetics
- Animals
- Antiviral Agents
- Base Sequence
- Cloning, Molecular
- DNA, Complementary/genetics
- Fish Proteins*/genetics
- Fish Proteins*/metabolism
- Gene Expression Regulation
- Humans
- Interferon-gamma*/genetics
- Interferon-gamma*/metabolism
- Nuclear Localization Signals/genetics
- Nucleotides
- Saccharomyces cerevisiae/genetics
- Zebrafish
- PubMed
- 36298859 Full text @ Viruses
Citation
Jiang, J., Gu, J., Zhan, A., Mao, M., Liu, Y., Wang, H., Mao, Y. (2022) Cloning, Exogenous Expression and Function Analysis of Interferon-γ from Gadus macrocephalus. Viruses. 14(10):.
Abstract
Interferon γ (IFN-γ) is now considered to be one of the key molecules in the regulation of innate and adaptive immunity. The function of IFN-γ is best described in humans, but less of IFN-γ in fish species has been described at protein level. In the present study, IFN-γ from Gadus macrocephalus (GmIFN-γ) has been examined in terms of bioinformatics, prokaryotic expression, yeast expression, antiviral activity and immune regulatory function. The cDNA of GmIFN-γ contains an open reading frame of 570 nucleotides, coding 189 amino acids. The mature protein contains a nuclear localization signal motif and an obvious IFN-γ signature sequence at the C-terminal. GmIFN-γ is very similar to that of Atlantic cod, with homology up to 89.89%, but less than 32% to other species. GmIFN-γ can be detected in the gills, spleen, intestine, brain and kidney. Interestingly, during early development, a strong signal of GmIFN-γ was not detected until 40 days post hatching. Prokaryotic expression plasmid pET-32a-GmIFN-γ was constructed, and the expression products in BL21 were confirmed by Mass Spectrometry. Meanwhile, the plasmid pGAPZA-GmIFN-γ with Myc tag was constructed and transmitted into Pichia pastoris yeast GS115, and the products were tested using Western blot. The purified GmIFN-γ from either BL21 or yeast has a strong antivirus (Spring viremia of carp virus) effect. The vector of pcDNA3.1-GmIFN-γ was expressed in EPC cell lines; high transcript levels of MHC class I chain-related protein A (MICA) gene were detected; and the exogenous GmIFN-γ protein could also induce MICA expression, indicating that GmIFN-γ could stimulate immune response. The yeast GS115 with GmIFN-γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish IFN-γ was upregulated significantly; however, genes of the interferon type-I signal pathway were not well stimulated.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping