Cloning And Expression In E.coli And Insect Cells Of Huainan Porcine Interleukin-2

Interleukin-2 (IL-2) is a cytokine secreted by activated T lymphocytes and has an important role in a number of in vivo immune responses. The important function of 1L2 is to induce T cell proliferation and differentiation and to promote helper T cells and NK cells to product cytokines. In this study, the Huainan Porcine interleukin-2 was cloned firstly and subcloned into prokaryote and eukaryotic expression vectors respectively. Then, the biological activities of expressed protein was researched preliminary. the main content is as follows:1. One pair of primers were designed according to porcine IL-2 gene sequences published in GenBank. The interleukin-2 cDNA of Huainan Pig was amplified by RT -PCR from total RNA extracted from spleen lymphocytes,which were cultivated and stimulated with 10μg/mL ConA invitro for 27 hours.Then the amplified cDNA was cloned into pGEM-T Easy vector and sequenced. The result suggested that The full length of the Huainan pig IL -2 was 516bp (ORF was 465 bp ) , encoding a peptide of 154 amino acids whose molecular is 17. 4 kD ,pI is 5. 47.,hydrophobic amino acid is 34% , hydrophilic amino acid is 34%,basic amino acid is 11% and acidic amino acid is 23%.The sequence was just the same as Genebank published. Compared of the nucleotides sequence of pig IL-2 with those of cat, cattle, chicken, dog, duck, goat, horse, human and house mouse, showed that the nucleotides homology were 83.7%,82.6%,28.2%,80.6%,29.6%,83.4%,81.3%,82.0%,and 61.5% respectively.2. To express Huainan Porcine IL-2 in E. coli and investigate the bioactivity of the recombinant protein. One pair of primers was designed and the Huainan Porcine IL-2 gene was subcloned into the prokaryotic expression vector of pGEX-6p-1 and transformed into BL21. After induced by IPTG, the expression of porcine IL-2 was detected by SDS-PAGE, the specificity and bioactivity of expressed protein was confirmed by Western blot and MTT. The result showed that the Huainan Porcine IL-2 gene could high express in E. coli, the expressed protein possess specificity and biological activities.3. To express porcine IL-2 in baculovirus-insect cells expression system and investigate the bioactivity of the recombinant protein, one pair of primers was designed, and the porcine IL-2 gene was amplified, and subcloned into the Baculovirus transfer vector of pFastBacDual and transformed into DH10Bac which contains a shuttle vector of Bacmid. After co-transfected the recombinant plasmid into insect cells, the expression of porcine IL-2 were detected by indirect immunofluorescence and SDS-PAGE, the specificity and bioactivity of expressed protein were confirmed by Western-blotting and MTT. The result showed that the porcine IL-2 gene could express in insect cells, the expressed protein possess specificity and biological activities. In summary, in this experiment, the Interleukin-2 gene of Huainan procine of Henan Local Breed was cloned and expressed in the prokaryotic expression system and Bac-to-Bac expression system respectively, and the biological activities of expressed protein were detected by MTT. The study paved the way of the development of new immunologic adjuvant.