Expression And Characteristic As Genetic Adjuvant Of Recombinant Human Interleukin-12 Eukaryotic Vector

Objective To identify the expression of recombinant human interleukin-12 (rIL-12) vector (pcDNA6-p70) and biological activity of the rIL-12 ex vivo;to investigate the characteristic as genetic adjuvant of pcDNA6-p70 in the experimental mouse.Methods The expression of rIL-12 in HEK 293 cells was measured by ELISA.. Biological effect of rIL-12 on PBMC from blood donors was estimated by the methods of cytokine follow cytometry (CFC), tetramer staining, NK activation, non-specific and specific lymphocyte proliferation. The experimental mouse (Kunming Mouse and Balb/c) were co-immunized with pcDNA6-p70 and recombinant adenovirus encoding pp65 from human cytomegalovirus (HCMV)(adeno-pp65) once two weeks in 8 weeks. The characteristic as genetic adjuvant of pcDNA6-p70 was evaluated according as the specific cellular immunity against HCMV by means of CFC, CTL, specific lymphocyte proliferation, IFN- γ release. Meanwhile, the security of pcDNA6-p70 in vivo was investigated.Result The expression of rIL-12 in HEK293 cells was as high as 517pg/ml (5 × 10~4 cells). Under the effect rIL-12, the frequency of CD4/IFN- γ and CD8/IFN-γ double positive cells to the stimulation of PHA (5.0;4.55) and HCMV (7.78;5.82) were much higher than that of no-rIL-12 control (PHA: 0.3,1.05;HCMV: 3.76,2.46), moreover, the frequency of N9V specific CD8+ cells (1.19% vs 0.98%), NK activation, non-specific and specific lymphocyte proliferation were also increased under the effect of rIL-12. The results of animal experiment showed that in group of co-immunization, the frequency of the specific CD4/IFN-γ (5.17%) and, CD8/IFN-γ (4.24%) double positive cells were higher than that the groups of signal immunization with adeno-pp65(2.92%;2.02%) and normal saline (NS) control (2.05%;1.95%);and NK activation, CTL activation , specific lymphocyte proliferation , the production of specific and non-specific IFN- γ were also higher in co-immunized group. The results of security proved that no difference were fund between experimental mouse and controls.Conclusion pcDNA6-p70 could be expressed expectedly in HEK293 cells. The rIL-12 exited perfected biological activity ex vivo. The satisfactory function of genetic adjuvant was proved from pcDNA6-p70 in vivo. Furthermore, it was security to use the pcDNA6-p70 in experimental mouse.