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Molecular cloning and expression of cynomolgus monkey interleukin-1beta cDNA
K Totsuka1, H Takakura, O Hashimoto
1Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan.
Insights
Researchers cloned cynomolgus monkey interleukin-1beta (IL-1beta) cDNA, finding 90% homology to human IL-1beta. The recombinant protein expressed in E. coli and insect cells demonstrated biological activity.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Interleukin-1beta (IL-1beta) is a key inflammatory cytokine.
- Understanding non-human primate cytokine homologs aids comparative immunology and disease modeling.
Purpose of the Study:
- To molecularly clone and characterize cynomolgus monkey IL-1beta (IL-1beta) cDNA.
- To express recombinant monkey IL-1beta in prokaryotic and eukaryotic systems.
- To confirm the biological activity of the expressed recombinant monkey IL-1beta.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify IL-1beta cDNA from stimulated splenocytes.
- Sequence analysis determined the protein's amino acid composition and homology to human IL-1beta.
- Recombinant expression was achieved in Escherichia coli (as a thioredoxin fusion protein) and insect cells (using a baculovirus vector).
- Western blot analysis and bioassays were performed to characterize the recombinant protein.
Main Results:
- Cynomolgus monkey IL-1beta cDNA encodes a 268-amino acid protein with 90% homology to human IL-1beta.
- Amino acid substitutions were primarily located in the leader sequences.
- Recombinant monkey IL-1beta expressed in both E. coli and insect cells reacted with anti-human IL-1beta antiserum.
- The recombinant protein exhibited biological activity in a standard bioassay.
Conclusions:
- The cloned cynomolgus monkey IL-1beta cDNA provides a valuable tool for immunological research.
- The successful expression and confirmation of biological activity enable further studies on monkey IL-1beta function and its role in disease models.
Abstract:
The cynomolgus monkey cDNA encoding interleukin-1beta (IL-1beta) was molecularly cloned by the reverse transcription polymerase chain reaction from a cDNA library of adherent splenocytes stimulated with lipopolysaccharides. The sequence analysis showed that the monkey IL-1beta cDNA encodes a protein of 268 amino acids and displays a high degree (90%) of homology with the human counterpart. Substitution of amino acids resides mainly in the leader sequences of IL-1beta when compared with those of human IL-1beta. This cloned monkey IL-1beta cDNA was used to express in Escherichia coli as a fusion protein with thioredoxin and in insect cells infected with a recombinant baculovirus after molecular modification where monkey IL-1beta signal sequences were placed prior to the mature sequences of IL-1beta for efficient secretion in insect cells. Recombinant monkey IL-1beta expressed in both systems was shown to react with rabbit antihuman IL-1beta antiserum by Western blot analysis and to have the biological activity of IL-1beta in a bioassay.
