Molecular Cloning, Expression In E.coli Of Human IL-15 CDNA And Purification Of Recombinant Protein

Interleukin 15 ( IL-15 ) is a newly discovered pleiotropic cytokine that shares biological activities with EL-2 and, like IL-2, is a member of the four-helix bundle cytokine family. It has been shown that IL-15 utilizes both the β and γ subunits of the IL-2 receptor for binding and signal transduction. However, IL-15 is expressed by a variety of cells, and has a specific high-affinity IL-15 receptor a which has a wide distribution in different cell types. Many studies have shown that IL-15 play a important role in the development and differentiation of NK cell, initiation of immune responses, and in tumor, intracellular infection and immune-mediated inflammatory diseases, etc.In this paper, human EL-15 cDNA fragment was cloned from LPS-stimulated human adherent peripheral blood mononuclear cells by RT-PCR and inserted into pBlueSkm plasmid, identified by restriction endonucleases and DNA sequencing. Recombinant plasmid pAQ-EL15 was constructed by inserting the Nde I/BamH I fragment of IL-15 cDNA from recombinant plasmid pBS-IL15 into Nde I/BamH I pAQ vector. In addition, the amplified IL-15 cDNA fragment from plasmid pBS-IL15 was digested with BamH I and cloned into EcoR I (filled in)/BamH I pBV220 vector. The recombinant plasmid pBVB-IL15 was constructed. E. coli DH5α were transformed with recombinant plasmids and cultured at 42℃ to induce the expression of the foreign genes. The results of SDS-PAGE showed that there was a band of about 13kD in all the cells containing recombinant plasmid pAQ-IL15 and pBVB-EL15, respectively accounting for 32.5%, 23.9% of the total bacterial protein. Computer analysis showed that there is a secondary structure in the translational initiation region and a long distance between SD sequence and initiation codon AUG in the EL-15 mRNA transcripts from recombinant plasmid pBVB-EL15. These structures may interfere with the translation of EL-15 mRNA. The EL-15 cDNA was then reamplified from plasmid pBS-EL15, using primer to generate a 5'-EcoR I site, and cloned into EcoR I/BamH I pBV220 vector. The secondary structure in the translational initiation region of EL-15 mRNA was decreased and the distance between SD and AUG was reduced to 6 bp. The expressed recombinant protein was up to about 33.3% of the total bacterial protein in E. coli DH5a. Dynamic analysis with cell containing...