Abstract
Interleukin-2 is a vital cytokine secreted by activated T lymphocytes, and plays important role in the regulation of cellular and humoral immunity of animals. In our experiment, IL2 cDNA of the Tibet Pig was first cloned by RT-PCR from ConA-stimulated lymphocytes in the blood and subcloned into pMD-18 T vector, which then was identified with endonuclease restriction. The sequencing result showed that Tibet pig IL-2 (TPIL-2) cDNA was 503 bp long (ORF was 465 bp) (Genbank accession number: AY 294018). The recombinant prokaryotic and eukaryotic expression plasmids of the cDNA were then constructed to analyse the ability to stimulate the proliferation of porcine lymphocytes in vitro. The recombinant porcine IL-2 expressed in the prokaryotic cells was found to be of 43 kDa molecular mass, which was consistent with a 17.4 kDa protein deduced from the IL-2 cDNA sequence (glutathione S-transferase molecular mass is 26 kDa); the recombinant protein in eukaryotic cells was confirmed by use of specific rabbit anti-porcine IL-2 serum in an ELISA. The bioactivity of TPIL-2 was detected through MTT colorimetry by stimulating the proliferation of pig ConA-stimulated blasts in vitro. The results indicate that the TPIL-2 significantly promoted the proliferation of ConA-stimulated blasts of pig. This confirms that IL-2 cDNA of the Tibet pig was successfully cloned and expressed in prokaryotic and eukaryotic cells, which lays the foundation for the the preparation of specific recombinant IL-2 protein and development of novel immune adjuvants to raise the immunity of pigs against various infectious pathogens and increase the immunoprotective efficacy of vaccines.
Similar content being viewed by others
Abbreviations
- ConA:
-
concanvalin A
- DMEM:
-
Dulbecco’ modified Eagle’ medium
- ELISA:
-
enzyme-linked immunosorbent assay
- GST:
-
glutathione S-transferase
- IPTG:
-
isopropyl-α-D-thioglatotopyrano-side
- IL-2:
-
interleukin 2
- α-MM:
-
α-methyl-α-D-mannoside
- MTT:
-
3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide
- PBS:
-
phosphate-buffered saline
- pGTPIL-2:
-
the prokaryotic expression plasmid
- SDS:
-
Sodium dodecyl sulphate
- SDS-PAGE:
-
SDS-polyacrylamide gel electrophoresis
- TMB:
-
3,3′,5,5′-tetramethylbenzidine
- VRTPIL-2:
-
the eukaryotic expression plasmid
- X-gal:
-
5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hui, L., Rong, G., Mei, W. et al. Molecular Cloning and Expression of IL2 cDNA from the Tibet Pig. Vet Res Commun 29, 395–405 (2005). https://doi.org/10.1007/s11259-005-9141-1
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11259-005-9141-1