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Isolation, nucleotide sequence and expression of a cDNA encoding feline granulocyte colony-stimulating factor
1Department of Veterinary Pathology, University of Glasgow, Bearsden Road, G61 1QH, UK. s.dunham@vet.gla.ac.uk
Cytokine
|August 11, 2001
Summary
Researchers cloned feline granulocyte colony-stimulating factor (fG-CSF) cDNA and expressed active recombinant fG-CSF. This protein demonstrated significant biological activity in cell line assays, paving the way for further feline immunology research.
Area of Science:
- Molecular Biology
- Immunology
- Biochemistry
Background:
- Granulocyte colony-stimulating factor (G-CSF) is crucial for neutrophil production and function.
- Understanding feline G-CSF (fG-CSF) is important for feline immunology and potential therapeutic applications.
Purpose of the Study:
- To clone the cDNA encoding feline granulocyte colony-stimulating factor (fG-CSF).
- To express and purify biologically active recombinant fG-CSF.
- To characterize the biological activity of recombinant fG-CSF.
Main Methods:
- Cloning of fG-CSF cDNA from feline alveolar macrophages using reverse transcriptase-polymerase chain reaction (RT-PCR).
- Expression of recombinant fG-CSF as a glutathione S-transferase (GST) fusion protein.
- Purification of the recombinant protein via affinity chromatography.
- Assessment of biological activity using the murine myeloblastic cell line GNFS-60.
Main Results:
- Successfully cloned a 949 bp cDNA encoding fG-CSF, predicting a mature protein of 174 amino acids.
- Produced and purified recombinant fG-CSF with demonstrated biological activity.
- Determined an ED50 of approximately 2 ng/ml for fG-CSF using the GNFS-60 cell line.
Conclusions:
- The successful cloning and expression of active recombinant fG-CSF provide a valuable tool for feline immunology research.
- The demonstrated biological activity confirms the functional integrity of the recombinant feline G-CSF protein.