Cloning, Exogenous Expression and Function Analysis of Interferon–γ from <i>Gadus macrocephalus</i>
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 <i>Gadus macroceph...
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MDPI AG
2022-10-01
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| Online Access: | https://www.mdpi.com/1999-4915/14/10/2304 |
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| author | Jielan Jiang Jie Gu Aijun Zhan Mingguang Mao Yumeng Liu Haishan Wang Yunxiang Mao |
| author_facet | Jielan Jiang Jie Gu Aijun Zhan Mingguang Mao Yumeng Liu Haishan Wang Yunxiang Mao |
| author_sort | Jielan Jiang |
| collection | DOAJ |
| description | 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 <i>Gadus macrocephalus</i> (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 <i>Pichia pastoris</i> 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 <i>pcDNA3.1</i>–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 <i>Gm</i>IFN–γ protein could also induce MICA expression, indicating that GmIFN–γ could stimulate immune response. The yeast GS115 with <i>Gm</i>IFN–γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish <i>IFN–γ</i> was upregulated significantly; however, genes of the interferon type–I signal pathway were not well stimulated. |
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| issn | 1999-4915 |
| language | English |
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| spelling | doaj.art-78e9333b9e2b4ad496c58a9a690b7d5a2023-11-24T03:11:08ZengMDPI AGViruses1999-49152022-10-011410230410.3390/v14102304Cloning, Exogenous Expression and Function Analysis of Interferon–γ from <i>Gadus macrocephalus</i>Jielan Jiang0Jie Gu1Aijun Zhan2Mingguang Mao3Yumeng Liu4Haishan Wang5Yunxiang Mao6Yazhou Bay Innovation Institute, Hainan Tropical Ocean University, Sanya 572022, ChinaCollege of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, ChinaShenzhen Academy of Inspection and Quarantine, Shenzhen 518010, ChinaYazhou Bay Innovation Institute, Hainan Tropical Ocean University, Sanya 572022, ChinaCollege of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, ChinaYazhou Bay Innovation Institute, Hainan Tropical Ocean University, Sanya 572022, ChinaYazhou Bay Innovation Institute, Hainan Tropical Ocean University, Sanya 572022, ChinaInterferon 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 <i>Gadus macrocephalus</i> (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 <i>Pichia pastoris</i> 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 <i>pcDNA3.1</i>–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 <i>Gm</i>IFN–γ protein could also induce MICA expression, indicating that GmIFN–γ could stimulate immune response. The yeast GS115 with <i>Gm</i>IFN–γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish <i>IFN–γ</i> was upregulated significantly; however, genes of the interferon type–I signal pathway were not well stimulated.https://www.mdpi.com/1999-4915/14/10/2304interferon γprokaryotic expressionyeast expression<i>Gadus macrocephalus</i> |
| spellingShingle | Jielan Jiang Jie Gu Aijun Zhan Mingguang Mao Yumeng Liu Haishan Wang Yunxiang Mao Cloning, Exogenous Expression and Function Analysis of Interferon–γ from <i>Gadus macrocephalus</i> Viruses interferon γ prokaryotic expression yeast expression <i>Gadus macrocephalus</i> |
| title | Cloning, Exogenous Expression and Function Analysis of Interferon–γ from <i>Gadus macrocephalus</i> |
| title_full | Cloning, Exogenous Expression and Function Analysis of Interferon–γ from <i>Gadus macrocephalus</i> |
| title_fullStr | Cloning, Exogenous Expression and Function Analysis of Interferon–γ from <i>Gadus macrocephalus</i> |
| title_full_unstemmed | Cloning, Exogenous Expression and Function Analysis of Interferon–γ from <i>Gadus macrocephalus</i> |
| title_short | Cloning, Exogenous Expression and Function Analysis of Interferon–γ from <i>Gadus macrocephalus</i> |
| title_sort | cloning exogenous expression and function analysis of interferon γ from i gadus macrocephalus i |
| topic | interferon γ prokaryotic expression yeast expression <i>Gadus macrocephalus</i> |
| url | https://www.mdpi.com/1999-4915/14/10/2304 |
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