Construction Of The Recombinant Of Hsv-2 Gd Minotope And Il-18 Dna Vaccines And Its Inducing Immune Respons In Mice

ObjectiveThe P6 is a mimotope of HSV-2 gD and may be a substitute antigen of gD. Find its similar sequence of natural HSV-2 gD from gene bank. To construct and express pcDNA3.1-IL-18-HSV P6. pcDNA3.1-IL-18-HSV NP6 and pcDNA3.1-IL-18 recombinant DNA vaccines then explore the immune response in mice induced by them.Methods(1)Genes of P6(NP6) and IL-18 were subcloned into the eukaryotic expression vectors pcDNA3.1(-) respectively. After confirmed by enzyme digestion and sequence analysis, the recombinant plasmids pcDNA3.1-IL-18-HSV P6, pcDNA-3.1-IL-18-HSV NP6 and pcDNA3.1-IL-18 were transformed into CHO cells by Lipofectamine2000. The expressed protein were characterized by indirect immunofluorescence assay and Western blotting; (2)The BALB/c mice were divided into six groups randomly and each group has ten mice. The different reagents were injected into bilateral tibialis anterior muscle in mice. Each group:1. NS 100μl,2. pcDNA3.1100μl(1mg/ml),3. pcDNA3.1-IL-18100μl(1 mg/ml), 4.Synthetical P6 peptide100μl(l mg/ml),5. pcDNA3.1-IL-18-HSV P6100μl(1 mg/ml),6. pcDNA3.1-IL-18-HSV NP6100μl(1 mg/ml). Immunity once a week, a total of three times. The first time the Freunds Adjuvant Com applied to enhance the immune effect. Blood were collected a week after the final immunization to detect the levels of HSV-2 specific IgG,IL-18 and IFN-y in serum by means of ELISA. Lympocyte transformation test (LTT) was made by using MTT in the first month after the last immunization.Results(1) Recombinant pcDNA3.1-IL-18-HSV P6,pcDNA-3.1-IL-18-HSV NP6 and pcDNA3.1-IL-18 plasmids were confirmed by enzyme digestion and DNA sequencing. Indirect immunofluorescence and Western blotting all indicated that the mimotope of HSV-2 gD P6 have the similar Biological activity as its similar sequence of natural HSV-2 gD NP6; (2) The results showed that mice immunized with recombinant clone pcDNA3.1-IL18-HSV P6 are best in the experiment groups both in humoral immunity and cellular immunity.Conclusion(1) We constructed pcDNA3.1-IL-18-HSV P6,pcDNA3.1-IL-18-HSV NP6,pcDNA3.1-IL-18 recombinant DNA vaccines successfully. (2)Results of immunity in mice suggest that the recombinant clone pcDNA3.1-IL18-HSV P6 can effectively induce humoral and cellular immune responses.