Study On The Immune Responses Induced By DNA Vaccine With The Pp65 Gene Of HCMV In Mice

Objective To observe the cellular and humoral immune responses induced by phosphoprotein 65 DNA vaccine of human cytomegalovirus in Balb/c mice.On the aim to demonstrate whether this kind of vaccine has Hyperimmunization and side effect of hypotoxicity.It is significant for preventing and curing HCMV infection,especially couple of gravida and reproductive life.In the same time ,for obviating disability children of nerve. pp65 DNA vaccine was made by pp65 antigen amplified from HCMV AD 169 strain and cloned into the eukaryotic vector.Methods Twenty-four female healthy Balb/c 6-8 weeks old mice were randomly divided into three groups(A, B, C).Each group include eight mice.In group A(blank control group):using 100ul normal saline and injected into Balb/c mice via musculus quadriceps fexoris of left side hind legs, the mice were immunized three times at 0, 2, 4 week; In group B(pp65 DNA vaccine group): using 200ug pp65 DNA vaccine and injected into Balb/c mice via musculus quadriceps fexoris of left side hind legs , the mice were immunized three times at 0, 2, 4 week; In group C(pp65 DNA vaccine+CPG ODN):Each mouse was immunized with 200ug pp65 DNA and 10ug adjuvant CPG ODN for three times at 0, 2, 4 week through musculus quadriceps fexoris of left side hind legs. The third day before immunization,the immune position of each mouse was injected 0.5mg/ml hydrochloric acid bupivacaine 50 ul for pre-disposal treatment.The third week after the first immunization, cutting off the tail of each mouse and collecting blood serum.The week after the last immunization,collecting blood through picking bulb of eye, some blood add 0.1ml EDTA Anticoagulation the others collect bloodserum.The means of indirect ELISA detect pp65 antibody titer of mice serum.Using the ways of indirect immunofluorescence(IF),applying flow cytometry(FCM) determinate CD3+- CD4+ and CD8+ T cell subgroup.Using the ways of double antibody sandwich ELISA detect EFN-y of mice serum.Using the ways of radio-immunity(RI) to detect IL-2 of mice serum.Result 1.After Balb,c mice were immunized by DNA vaccine and pp65 DNA + CPG ODN,antibody titer of blood serum were tested:the titer were < 1:10, 1:11.89, 1:25.94 in group A,B,C respectively in the third week.the titer of anti-pp65 antibody were 1:95.14 in group B and 1:207.49 in group C.One week after the last immunization,T-Lymphocyte Subgroup (CD4+ , CD8+, CD3+) were tested in mice serum,The result of CD3+ was 54.94 4.06 in group A, 65.04 2.33 in group B and 74.16 2.08 in group C. The result of CD4+ was 29.73 1.83 in group A, 42.72 1.14 in group B and 48.44 1.66 in group C.The result of CD8+ was 18.82 1.09 in group A, 23.15 1.43 in group B and 26.44 1.37 in group C. The diference of interclass has statistical significanc (P<0.01) .3. cytokine IFN-yof mice serum was 98.5 9.86 in group A, 165.17 17.56 in group B and 245.50 26.29 in group C. The diference of interclass has statistical significanc (P<0.01) .4. cytokine IL-2 of mice serum was 5.52 0.73 in group A, 8.56 0.90 in group B and 14.09 1.50 in group C. The diference of interclass has statistical significanc (P<0.01) .conclusion 1. phosphoprotein 65 DNA vaccine of human cytomegalovirus can induce the cellular and humoral-mediated special immune responses in Balb,c mice. 2.immunological adjuvant CpG ODN can enhance immunity of phosphoprotein 65 DNA vaccine of human cytomegalovirus in Balb,c mice.