Cloning, Expression And Bioactivity Analysis Of Interleukin-2 Gene Of Tibet Pig

The interleukin-2 cDNA of Tibet Pig was cloned by RT-PCR from total RNA extracted from blood lymphocytes, which were cultured in RPMI 1640 medium and stimulated with 10ug/ml ConA in vitro for 70 hours. The interleukin-2 cDNA of Tibet pig was then subcloned into pUC18 vector and was named as TPIL-2. The nucleotide acid sequence of cloned Tibet pig interleukin-2 cDNA was determined. The length of the TPIL-2 was 503 bp (ORF was 465 bp), encoding a peptide of 154 amino acids whose molecular weight is 17.4 KDa and pi is 5.38. By blasting the homologous sequences in GenBank database, the sequence of Tibetan pig interleukin-2 gene from lymphocyte is identical to the interleukin-2 genes previously cloned from spleen cell and fibroblast cell of other porcines.The TPIL-2 cDNA fragment was inserted into pGEX-4T-l plasmid to construct the expression plasmid pGTPIL-2, the recombinant plasmid was digested by BamH I and EcoR I to identify whether theTPIL-2 cDNA fragment was inserted into the plasmid in correct orientation. The recombinant pGTPIL-2 was then transformed into E.coli DH5ct competent cells. The mRNA and protein expression the recombinant wereassayed by RT-PCR and SDS-PAGE. The results were found that the specific 465bp DNA bases of IL-2 was detected by RT-PCR in the recombinant bacteria and a new protein band was found in SDS-PAGE with molecular mass of about 43.4KDa, which is consisted of a 17.4 KDa protein of the TPIL-2 gene and GST (26 KDa). It indicates that the Tibet porcine IL-2 gene was correctly transcribed and translated in the transformed bacteria. The fusion protein was recovered from polyacrylamid gel and used to immunize the rabbit, and the titer of rabbit anti-porcine TPIL-2 serum from immunized rabbit was 1: 51,200, which could be employed in the detection of content and the bioactivity analysis of the porcine IL-2 in vitro and in vivo. These results proved that the Tibet porcine IL-2 gene and its recombinant protein could be further applied to develop effective immunoadjuvants to enhance the immunity of animals against infectious diseases.