Related Experiment Videos
Biological functions of recombinant bovine interleukin 6 expressed in a baculovirus system
M Yoshioka1, Y Mori, S Miyazaki
1National Institute of Animal Health, Tsukuba, Japan. miyako@niah.affrc.go.jp
Insights
Researchers cloned bovine interleukin 6 (IL-6) and found it affects liver protein production. However, bovine IL-6 did not stimulate mouse cells, indicating a species-specific interaction.
Area of Science:
- Molecular Biology
- Immunology
- Veterinary Science
Background:
- Interleukin 6 (IL-6) is a key cytokine involved in immune responses and inflammation.
- Understanding species-specific cytokine functions is crucial for comparative immunology and disease research.
Purpose of the Study:
- To clone and express functional bovine interleukin 6 (IL-6).
- To investigate the biological activity of recombinant bovine IL-6 on primary bovine hepatocytes.
- To assess the species-specific interactions of bovine IL-6 with cell lines.
Main Methods:
- Messenger RNA (mRNA) from lipopolysaccharide-stimulated bovine Kupffer cells was used for reverse transcription polymerase chain reaction (RT/PCR) to obtain bovine IL-6 cDNA.
- The cDNA was cloned into a baculovirus vector (pVL 1392) for expression in insect cells (Spodoptera frugiperda Sf21AE).
- Recombinant protein activity was tested on primary bovine hepatocytes and a murine IL-6-dependent cell line (7TD1).
Main Results:
- Recombinant bovine IL-6 was successfully expressed and secreted by insect cells.
- The secreted protein increased haptoglobin and decreased albumin production in bovine hepatocytes.
- Bovine IL-6 did not stimulate the proliferation of 7TD1 cells, unlike swine and human IL-6, suggesting species restriction.
Conclusions:
- Functional bovine IL-6 was successfully produced using a baculovirus expression system.
- Bovine IL-6 exhibits specific biological activities on bovine hepatocytes, affecting acute-phase protein synthesis.
- A significant species restriction exists between bovine IL-6 and murine IL-6-dependent cell lines.
Abstract:
The cDNA encoding bovine interleukin 6 (IL-6) was obtained from messenger RNA extracted from lipopolysaccharide-stimulated bovine Kupffer cells by the reverse transcription polymerase chain reaction (RT/PCR), and cloned into the baculovirus vector pVL 1392. Insect cells (Sf21AE derived from Spodoptera frugiperda) infected with the recombinant baculovirus secreted a large amount of 23.7 kD protein into the culture medium. This protein was capable of causing increased haptoglobin production and decreased albumin production in primary cultured bovine hepatocytes. The swine and human IL-6s were also able to decrease albumin production in bovine hepatocytes. This recombinant IL-6 did not stimulate the proliferation of 7TD1 cells (a murine IL-6-dependent cell line), whereas the recombinant swine IL-6 which was expressed in the same baculovirus system, and recombinant human IL-6 derived from Escherichia coli were each capable of stimulating proliferation of 7TD1 cells, respectively. This suggests a species restriction between bovine IL-6 and murine IL-6 dependent cell lines.