Cloning Of Mature Human Interleukin-15 CDNA And Expression Of The Protein In Bel-7402 Cells And Immune Adjuvant Effect On HBsAg DNA Vaccine

Objective To construct the Eukaryotic Expression Vector with human Interleukin-15 (IL-15) cDNA and to express in Bel-7402 Cells, which would benefit to the further research in the biological activities and the clinical applications of IL-15. Meanwhile to observe the immune responses to HBsAg DNA vaccine (pcDNA3.1(+)-S) and eukaryotic expression vector with IL-15 (pcDNA3.1hisB-IL-15) in BALB/c mice. Methods The entire human Interleukin-15 (IL-15) coding region, deleted of all the uncoding fragments, was amplified from human peripheral blood mononuclear cells by RT-PCR and cloned into pGEM -T Easy Vector; then, subcloned into the BamH I and EcoR I sites of the pcDNA3.1hisB plasmid, forming the recombinant eukaryotic expressing vector, pcDNA3.1hisB-IL-15, which was identified by the BamH I and EcoR I enzymatic digestion, PCR amplification and the sequence analysis. The construct was transfected into BEL-7402 cells by means of lipofectamine plus?Reagent, followed by a series of determinations to screen the positive cell colonies, such as RT-PCR, SDS-PAGE, ELISA and CTLL-2 proliferation assay. Meanwhile BALB/c mice were immunized by intramuscular injections with pcDNA3.1(+)-S orcoimmunized with pcDNA3.1hisB-IL-15. Six weeks after immunization, the anti-HBs levels in sera of mice and specific cytokine levels in supernatant of splenocytes cultured with ConA were detected by ELISA, HBsAg-specific lymphocyte proliferation test were assyed in vitro, NK and specific CTL activities were measured by LDH method. Results Sequence analysis verified that the fragment cloned in pcDNA3.1hisB was hIL-15 cDNA, 6 positive cell colonies highly expressed human IL-15 were obtained after G418 selection. On an average, the IL-15 protein content of supernatant from the BEL-7402-IL-15 cells was 2.86+0.613 ug/ml, the bioassay showed the activities were 156-252 U/(ml.l06cell.d). The serum 450nm A values of single immunization with pcDNA3.1(+)-S and the co-immunization with pcDNA3.1hisB-IL-15 were 1.89+0.62 and 1.37 + 0.58 respectively. The NK activities were 30.04+2.91 and 44.56+5.69 respectively, there was significant difference(P<0.05). Compared with mice injected with pcDNA3.1(+)-S alone, the specific CTL cytotoxity activity of mice co-immuned with pcDNA3.1hisB-IL-15 was significantly enhanced (P<0.05). The level of IFN- y in supernatant of splenocytes was significantly elevated (P<0.05) while the level of IL-4 did not chang significantly (P>0.05). The stimulatory index (SI) of splenocytes stimulated with HBsAg in co-immunized group was significantly elevated and had a dose-dependent increase.Conclusions Eukaryotic expression vector with human IL-15cDNA was constructed successfully and human IL-15cDNA was expressed efficiently in BEL-7402 cells. The plasmid of pcDNA3.1hisB-IL-15 together with HBsAg DNA vaccine may enhance cellular immune response elicited in mice.