Molecular Cloning and Expression of IL2 cDNA from the Tibet Pig |
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Authors: | Li Hui Gao Rong Wu Mei Wang Li-Huan Fu Man-Liang Wu Kai-Yuan |
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Institution: | (1) Key Lab of Biological Resource and Ecological Enviroment of Education Ministry, Life Science College, Sichuan University, Chengdu, Sichuan, 610064, PR China |
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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. |
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Keywords: | bioactivity assay cloning eukaryotic expression IL2 cDNA of Tibet pig prokaryotic expression |
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