Molecular cloning and expression of IL2 cDNA from the Tibet pig.
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.
Li
Hui
H
Key Lab of Biological Resource and Ecological Enviroment of Education Ministry, Life Science College, Sichuan University, Chengdu, Sichuan, PR China.
Gao
Rong
R
Wu
Mei
M
Wang
Li-Huan
LH
Fu
Man-Liang
ML
Wu
Kai-Yuan
KY
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology*; D007376:Interleukin-2 / Q000235:genetics* / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D013552:Swine / Q000378:metabolism*; D018609:Tibet
D018076:DNA, Complementary; D007376:Interleukin-2; D012313:RNA