| To construct the multigenic DNA vaccine of glycoproteinD(gD),glycoprotein B(gB),glycoprotein C(gC), infectious cell protein 27(ICP 27) , which then were modified by signal peptide of glycoproteinD(gD) and ubiquitin, and to investigate the humoral immunity andcell-mediated immunity induced by the multigenic DNA vaccine andcoimmunization with interlcukin-2 genetic adjuvantin micc.MethodsHSV-2 ICP27 377-459,gD146-179,gD223-306,gB529-606, gC247-282,gD1-77 gene sequences obtained by PCR amplification from domestic HSV-2 wild strain , multiple DNA fragments were cloned into pGEMT vector by one step quickly cloning method with using Isoschizomer-heterotailrestriction Endonuclease (IHRE), and the products of the fused aim gene were identified. HV was then directly cloned into eukaryotic expression vector pcDNA3．1,and got recombinant vector HV-pcDNA3．1 that was comfirmed by the restriction endonuclease and DNA sequencing.Signal peptide of gD was obtained by PCR amplification and got recombinant vector gDs-HV-his-pcDNA3．1 by endonuclease and DNA sequencing.Human ubiquitin and HSV-2 ICP27 377-459 gene sequences were obtained by PCR amplification , then be fused. The fused gene then was cloned into eukaryotic expression vector pcDNA3．1, and got recombinant vector Ub-ICP pcDNA3．1 that was comfirmed by the restriction endonuclease and DNAsequencing.Signal peptide and terminal gene sequences of human IL2 were obtained by PCR amplification , then be fused. The fused gene of IL2-cDNA, then was cloned into eukaryotic expression vector pcDNA3．1, and got recombinant vector IL-2-pcDNA3．1 that was comfirmed by the restriction endonuclease and DNA sequencing.The C57/BL6 mice were divided into eight groups randomly and each group have five mice. The former six groups were inoculated subcutaneouly by the pcDNA3．1 , HV-pcDNA3．1, HV-pcDNA3．1 + IL2-pcDNA3．1, gDs-HV-his-pcDNA3．1,gDs-HV-his-pcDNA3．1 + IL2-pcDNA3．1 and Ub-ICP-pcDNA3．1 , another two groups were inoculated intramuscularly by the HV-pcDNA3．1 and HV-pcDNA3．1 + IL2-pcDNA3．1 respectively.Each 5ug plasmid vectors and lug lipidosome were mixed and transfected into the splenic lymphocyte of female C57/BL6 mice , then cultivated for four hours. The former six groups were injected the specific antigen subcutaneously , another two groups were injected the specific antigen intramuscularly at first, and boostered after 14 days and 28 days by each 100ug plasmid vectors and 10ug lipidosome.Blood were collected 3 weeks after the final immunization to detect the levels of HSV-2 specific IgG, IL-2 and IFN-γin serum by using ELISA. The function of CTL was detected by LDH assay, the transformation of CD4+/CD8+ T lymphocyte was detected by flow cytometry.ResultsRistriction endonuclease digestion analysis of the recombinant vector HV-pcDNA3．1, IL2-pcDNA3．1, Ub-ICP-pcDNA3．1 and gDs-HV-his-pcDNA3．1 showed that were is identical with aim gene fragments; DNA sequence analysis showed the correct orientation and the same homology.HV-pcDNA3．1 remarkably enhanced the levels of the specific IgG, IL-2, IFN-γand CTL activity when compared with pcDNA3．1．The most effective humoral immune response in mice were induced by coimmunization with gDs-HV-his-pcDNA3．1 andIL2-pcDNA3, the valence of HSV-2 specific IgG was increased to 400．The most celluar immune response in mice were induced by coimmunization with HV-pcDNA3．1 and IL2-pcDNA3．1, the specific T lymphocytic stimulation index was 2．75 compared with 0．7 induced by pcDNA3．1, and the percentage of CTL activity was about 50% compared with 8% induced by pcDNA3．1;the level of IL2 was 1421．16±220．98 ng/L compared with 685．21±104．20 ng/L induced by pcDNA3．1 and the level of IFN-γwas 1956．19±219．60 compared with 547．71±189．33 ng/L induced by pcDNA3．1．The liposome-coated DNA vaccine enhanced the levels of the specificTgG,IL-2,IFN-γand CTL activity when compared with naked plasmid.Themultigenic DNA vaccine coimmunization with IL-2 cDNA can enhance moreeffective immune response .Conclusions1．The gD, gB, gC, TCP 27, multigenic DNA vaccines has been successfullyconstructed and modified by signal peptide of glycoprotein D(gD) andubiquitin , as well as the interleukin-2 genetic adjuvant IL2-pcDNA3．1．2．The multigenic DNA vaccine HV-pcDNA3．1 can induce effective humoral and cellular immune response in mice, and coimmunization with IL-2 cDNA can enhance more effective immune response . The most effective humoral immune response in mice were induced by coimmunization with gDs-HV-his-pcDNA3．1 andIL2-pcDNA3．The most celluar immune response in mice were induced by coimmunization with HV-pcDNA3．1 and IL2-pcDNA3．1．3．Inoculation subcutaneouly by the liposome-coated plasmid remarkably enhanced the immunological competence of multigenic DNA vaccine when compared with naked plasmid.4．Ub-ICP-pcDNA3．1 can not induce effective humoral and cellular immune response in mice.
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