Cloning And Expression Interleukin-2 Gene Of Rongchang Porcine

Based on the published nucleotide sequence of porcine familiary IL-2 gene ( X58428 ) , apair of RT-PCR primers was designed and synthesized. Total RNA, isolated from ConA-stimulated peripheral blood T cells of Rongchang porcine, was used as template to generate complementary DNA by reverse transcription. The 520bp DNA fragment was amplified by polymerase chain reaction, and cloned into PMD18-T vector. DNA sequencing confirmed the inserted fragment was porcine IL-2. By blasting the homologous sequences in Genbank databases, the sequence of Rongchang pig interleukin-2 gene from lymphocyte is 99.8% percent identity to the interleukin-2 genes previously cloned from other porcines.By the technology of DNA recombination, the IL-2 cDNA was cloned into expression plasmid PET-32(a)+ , and was transformed to Ecoli BL21. The recombinant bacteria was induced to expression through the changes of IPTG. The results were found that the specific 522bp cDNA 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 37KDa, which is consisted of a 17KDa protein of the IL-2 gene and PET-32(a)+ (20KDa). It indicates that the Rongchang porcine IL-2 gene was correctly transcribed and translated in the transformed bacteria. The biological activity of proteins was detected with lymphocyte transform assays. The results indicated that the recombinant porcine IL-2 proteins have the high ability to supply the proliferation of...