Construction And Expression Of Human IL-15in K562Cells

The gene of human interleukin-15(IL-15) located in the4q31, which is a34kbDNA sequences, with nine exons and eight introns. The whole length of human IL-15cDNA sequences was acquired from human bone marrow stromal cell lines by genetagging with the monkey source gene used as probes. The whole length of IL-15cDNA is1020bp, including a316bp length5’nontranslational section, a486bplength open reading frame and a400bp length3’nontranslational terminus. Thepreview polypeptide of IL-15contains162amino acids, after the signal peptide beencleaved, it transforms into mature IL-15, which is composed of114amino acids. ThepI of IL-15is4.35and the molecular mass is14~15KD.Human interleukin-15is a multifunctional cytokine, belongs to the four-helixbundle cytokine family. IL-15shares with IL-2the IL-2receptor (IL-2Rβ/IL-2Rγ),which contributes it the similar biological function with IL-2. IL-15almost canreplace the effect of IL-2plays in immune system such as to promote the proliferationand differentiation of lymphocyte, to induce natural killer cells to produce IFN-γ orTNF-α and to induce CTL or LAK activity. Furthermore, by using murine models, ithas been shown that IL-15induces a higher cytotoxicity compared with IL-2, and thedose of IL-15required to induce vascular leakage syndrome is six times higher thanthat of IL-2required. All those show that the therapeutic index of IL-15is superior toIL-2. Because of the unique function and advantages, IL-15has a broad applicationprospects in antitumor biological treatment.In this study, we separately built a secretory type of human IL-15and amembrane-binding human IL-15type by gene cloning and sub cloning. The two kindsof reconstructed expression vectors were utilized to transfect K562tumor cells, and astably expressed membrane-binding IL-15K562cell line was acquired. At the sametime, the expression of secretory human IL-15in K562cells was indentified. Thoseresearches would afford a foundation for the clinical application of IL-15and its genetherapy for tumor disease. The main work and results of this study are summarizedbelow: 1. Construction of the secretory IL-15expression vectorThe DNA encoding IL-15was obtained by RT-PCR with the RNA derived fromhuman peripheral blood mononuclear cells (PBMCs) as RT-PCR template. Then thecDNA was utilized as a new template for the PCR of IL-15CDS sequences with twoenzymes cutting sites of SalⅠand BamHⅠat the two ends. The PCR products wereclone into pMD18-T vector, after sequencing a correct gene IL-15was sub clonedinto pVITRO2-neo-mcs by SalⅠand BamHⅠ double digestion and a secretoryhuman IL-15expression vector pVITRO2-IL-15was constructed.2. Construction of the membrane-binding IL-15expression vectorWith the coding sequence of IL-15as template, the DNA of membrane-bindingIL-15was synthesized by overlapping PCR and the CD8α transmembrane peptidewas added into the N terminal of PCR products. By gene cloning and sub cloning, amembrane-binding human IL-15expression vector pMD18-T-mbIL-15wasconstructed. And the sequencing results demonstrated the result was correct.3. Expression of recombinant IL-15in K562cellsWe transfected expression vectors pVITRO2-IL-15and pVITRO2-mbIL-15intoK562cells respectively to detect the expression of secretory IL-15in K562cells andscreen K562cells which could stably express membrane-binding IL-15by flowcytometry.In conclusion, human IL-15DNA was successfully amplified by RT-PCR frommRNA that was extracted from human peripheral blood mononuclear cells. Then weobtained the coding sequence of IL-15and the sequence of membrane-binding IL-15.After sequencing, two correct expression vectors pVITRO2-IL-15and pVITRO2-mbIL-15were transfected into K562cells. We detected secretory IL-15in K562cellsby western blot, obtained stable expression of membrane-binding IL-15K562cells.