Abstract
Abstract
IFN-γ is a major effector cytokine, produced to induce type I immune responses. It has been cloned in several fish species including zebrafish, however to date few studies have looked at IFN-γ protein expression and bioactivity in fish. Hence, the current study focused on developing a monoclonal antibody (moAb) against zfIFN-γ. We show that the zfIFN-γ moAb specifically recognises E. coli produced recombinant IFN-γ protein and zfIFN-γ produced in transfected HEK293 cells, by Western blot analysis. Next we analysed the production of the native protein following expression induced by PHA stimulation of leukocytes in vitro or antigen re-stimulation in vivo. We show the IFN-γ protein is produced as a dimer, and that a good correlation exists between transcript expression levels and protein levels.
IFN-γ is a major effector cytokine, produced to induce type I immune responses. It has been cloned in several fish species including zebrafish, however to date few studies have looked at IFN-γ protein expression and bioactivity in fish. Hence, the current study focused on developing a monoclonal antibody (moAb) against zfIFN-γ. We show that the zfIFN-γ moAb specifically recognises E. coli produced recombinant IFN-γ protein and zfIFN-γ produced in transfected HEK293 cells, by Western blot analysis. Next we analysed the production of the native protein following expression induced by PHA stimulation of leukocytes in vitro or antigen re-stimulation in vivo. We show the IFN-γ protein is produced as a dimer, and that a good correlation exists between transcript expression levels and protein levels.
| Original language | English |
|---|---|
| Pages (from-to) | 79-86 |
| Number of pages | 8 |
| Journal | Fish & Shellfish Immunology |
| Volume | 79-86 |
| Issue number | 57 |
| Early online date | 15 Aug 2016 |
| DOIs | |
| Publication status | Published - Oct 2016 |
Bibliographical note
AcknowledgmentsSY was supported by the Scottish Overseas Research Scholarship Awards Scheme (SORSAS) to undertake a PhD program at the University of Aberdeen. PTL was supported by a PhD studentship from the Ministry of Education, Republic of China (Taiwan). TYW was supported by grants from the Ministry of Science and Technology, Taiwan (NSC 100-2313-B-006-002-MY3 and MOST 103-2313-B-006-004-MY3) to visit Aberdeen. This research was also funded by the European Commission under the 7th Framework Programme for Research and Technological Development (FP7) of the European Union (grant agreement No. 311993 TARGETFISH).
Keywords
- Zebrafish
- IFN-γ
- Monoclonal antibody
- Protein expression
- PHA
- Antigen re-stimulation