| Objective: To observe the humoral and cellular immune response in Balb/c mice, besides factors which effected on DNA immunization were explored and to study the immune protection for preventing the congenital infection of HCMV in SPF female mice, the specific pp65 protein segment was expressed in E.coli BL21 and SP2/0 cells, and prepared the pp65 protein vaccine and pp65 DNA vacine molecular. On the aim to look for a new way to prevent maternal primary HCMV infection and then to avoid the congenital infection transmitted from mother.Methods:1. Predominant epitope coding region of pp65 (1006-1521nt) antigen was amplified from HCMV DNA by PCR, and cloned into the prokaryotic vector pET100-TOPO and eukaryotic vector pcDNA3.1-TOPO, the insert gene was identified by PCR and DNA sequencing, then expressed in E.coli BL21 bacteria and SP2/0 cells. The two recombinant proteins were identified with specific pp65 McAb by Western blotting, and purified the aim protein from including body and extracted the plasmid pcDNA3.1-pp65.2. To observe the humoral and cellular immune response in Balb/c mice, thirty-sixfemale Blab/c 6-8 weeks old mice were divided into six groups (A, B, C, D, E> F), In group A(pp65 protein vaccine group): each mouse was first immunized with 50ul pp65 purified pp65 protein (lug/ul)+50ul CFA by subcutaneous and boosted immunized with 50 l pp65 protein+50 lIFA by subcutaneous for two times after 2, 4 week. In group B (pp65 DNA vaccine group): Each mouse was immunized with 200ug pcDNA-3.1-pp65 plasmid DNA by intramuscular (i.m.)for three times at week 0, 2, 4. In group C(pp65 DNA vaccine +pp65 protein vaccine co-immunization group): each mouse was immunized (i.m.)with 200ug pcDNA-3.1-pp65 plasmid DNA for two times at week (K 2 and immunized with by subcutaneous with pp65 protein+50 lCFA after 4 week. In group D (pp65 DNA vaccine + CpG): each mouse was immunized (i.m.) with 200ug pcDNA-3.1-pp65 plasmid DNA and 20ug CpG for three times at week 0, 2, 4. In group E (plasmid negative control group): Each mouse was immunized (i.m.) with 200 g pcDNA-3.1 plasmid DNA for three times at week 0, 2, 4; In group F (blank control group): each mouse was immunized (i.m.) with 100ul Saline for three times at 0, 2, 4 week. Three days after last immunization, the spleens were collected and stimulated by recombinant pp65 antigen segment. The proliferation response of spleen cells and specific cytotoxic T lymphocyte (CTL) activity was tested by MTT assay. IFN- Y was detected by ELISA. At the same time, anti-HCMV pp65 antibodies were detected by ELISA assays.3.To Study the immune protection of pp65 protein vaccine and DNA vaccine on preventing the congenital infection of HCMV, twenty four SPF Balb/c mice (6-8 weeks old) were divided into four groups (half of mice are female). Every mouse in group A was injected into lOOul Saline, after a week, they were subjected to the intraperitoneum injection of 1.0 ml HCMV supernatant (100 TCID50), every mouse in group B and group C was immunized (i.m.) with 200ug pcDNA-3.1-pp65 plasmid DNA before one week of administering HCMV(100 TCID50), every mouse in group B and group C was given a booster immunization with 200ug pcDNA-3.1-pp65 plasmid DNA by i.m on the7th days of gestation-day,every mouse in group B and group C was given a booster immunization with 200 g pcDNA-3.1-pp65 plasmid DNA and 50 g pp65 protein respectively by i.m and subcutaneous on the 15th days of gestation-day. Group D was set up as normal control. After injection of HCMV, these mice were paired to match. Fetuses before delivery were removed from uteri. After the development state of the fetuses was observed, the cerebral cortex of the fetuses was removed from the skull. At the same time, the serum and spleen cells of female mice were collected in order to detect the humoral and cellular immune response of female mice: (1) HCMV virus was isolated from the supernatant of fetal mouse brain homogenate. (2) The histopathological and ultrastructual changes of the neurons of fetal mice fro...
|
PDF Full Text Request