Cloning And Prokaryotic Expression Study Of Interleukin-4 Gene Of Tibet Pig

Interleukin-4 cDNA from lymphocyte was synthesized by RT-PCR from mRNA extracted from blood lymphocytes of Tibet Pig, which were stimulated by 10ug/ml ConA in vitro for 70 hours. The porcine interleukin-4 cDNA from lymphocyte was then subcloned into pMDl 8-T vector and was named as pUTPIL-4. The sequence of cloned porcine interleukin-4 cDNA was determined. The length of the IL-4 gene was 440 bp (ORF was 402 bp), encoding a peptide of 134 amino acids whose molecular weight is 15 KDa and pi is 8.24. By blasting the homologous sequences in GenBank databases, The cloned Tibet pig interleukin-4 was 99 % homologus to the necleotide acids of Large White porcine and 98 % homologus to that of hybrid of Landrance with Chuanbai pig. It indicated IL-4 of Tibet pig was successesfully cloned from the lymphocyte after stimulating 70h with ConA.The TPIL-4 cDNA fragment was inserted into pGEX4T-l plasmid to construct the expression plasmid pGTPIL-4, the recombinant plasmid was digested by BamH I, EcoR. I and PmaC I to identify whether the TPEL-4 cDNA fragment was inserted into the plasmid in correct orientation, the pGTPIL-4 was transformed into Kcoli BL21 competent cells. The mRNA and protein expression were assayed by RT-PCR and SDS-PAGE. The results were found that the specific 440bp DNA bases of IL-4 was detected by RT-PCR in the recombinant bacteria and a new protein band was found in SDS-PAGE with molecular mass of about 41 KDa which is consisted of a 15 KDa protein deduced from the IL-4 gene sequence and GST (26 KDa). It indicated that the porcine IL-4 gene was correctly transcribed and translated in the recombinant bacteria The fusion protein was recovered from polyacrylamid gel and used to immunize the rabbit, and the titer of rabbit anti-porcine IL-4 serum from immunized rabbit was 1: 12,800,which could be employed in the detection of content and the bioactivity analysis of the porcine IL-4 in vitro and in vivo.