Cloning Of Mouse Interleukin 21 CDNA And Its Application In Tumor Gene Therapy

Objective To clone murine interleukin 21 (mIL-21) gene, constructing its eukaryocytic expression plasmid and applying it in murine Sp2/0 subcutaneous tumor model with gene therapy to evaluate its anti-tumor effect and possible mechanisms.Methods mIL-21 cDNA is amplified from Con A activated murine T cells by RT-PCR, and then the cDNA is cloned into eukaryocytic expression plasmid pcDNA3.1 to form recombinant plasmid pcDNA3.1/mIL-21. The recombinant plasmid is transfected into Sp2/0 via lipofectin after identification by restriction enzyme digesting and DNA sequencing. The expression of mIL-21 is detected by RT-PCR and Western Blot. The activity of the protein is identified according to its effects on either the stimulated co-proliferation of mouse T cells or the enhanced cytotoxicity of NK, which are measured by H3-TdR and MTT colorimetry respectively. After establishing tumor animal model by inoculating Sp2/0 s.c into BALB/c mice, the recombinant plasmid is injected directly into lump tissue in vivo. Tumor formation rate, tumor weight, the activities of CTL, NK and LAK cells, and the levels of antibody and cytokines in the serum of all mice are measured for evaluating the anti-tumor effect of mIL-21 and its possible mechanisms.Results The mIL-21 cDNA is successfully amplified from the Con A activated murine T cells. Though there are three bases difference between the sequence acquired by us and the sequence of GenBank accession number AF254070, the amino acid sequence coded proteins has no change. Our sequence has been accepted by GenBank with the accession number AY428162. The recombinant plasmid pcDNA3.1/mIL-21 is constructed correctly and the expression of gene and protein of mIL-21 is detected in transformed sp2/0 cells. The expression protein can co-stimulate the murine T cells proliferating and enhance the cytotoxicity of NK cells in vitro. The subcutaneous tumor model in mouse is established successfully. After treatment with the recombinant plasmid, the tumor growth is suppressed; the activities of immune cells are improved dramatically, such as CTL and NK. The level of IFN-? is higher than that of control mice, but no differences is found in the LAK activity and antibody level between experiment and control mice.Conclusion The cloning of mIL-21 cDNA is successful and the recombinant plasmid pcDNA3.1/mIL-21 has also been constructed successfully, which can express mIL-21 protein with bio-activity. The gene therapy of mIL-21 can suppress the tumor growth in murine tumor model. The activity of the cell-mediated immunity in tumor-bearing mice is improved dramatically, especially NK and CTL activities. These results suggested that pcDNA3.1/mIL-21 inoculated directly into tumor is a potential approach for gene therapy and its mechanism may associate with the cell mediated immunity against tumor cells.