| Aim: Our studies were based on oral administration of Lactococcus lactis expressing recombinant P30 of Toxoplasma gondii (L.lactis/P30) which had been successfully constructed. 1. In part I of this study, the immunoproctive effect of oral administration of L.lactis /P30 in mice would be evaluated. First of all, the optical immuning dose for L.lactis /P30 orally immunizated with mice would be determined. Under the selected immuning dose, the humoral immune response and the protective effect against challenge in mice would be observed. Then, the cellular response and the kinetics of development of antibody IgG would be studied. At last, the change of IFN- , IL-2 and NO in the sera from the mice orally immunized with L.lactis /P30 would be observed.2. Part II of this study would be focused on multiple antigenic complex vaccine for toxoplasmosis in order to enhance the immunoprotective effect of L.lactis /P30 as oral administration. The genes coding for rhoptry protein ROP2 and major surface protein P30 from the total DNA of Toxoplasma gondii of RH strain would be cloned into plasmid pET28b.The recombinant plasmid of pET28b-ROP2-P30 would be transformed into BL21-Codon Plus(DE3)-RIL strain cells of E.coli , and expression products were going to be analysed for their antigenicity. Our study would offer the possibility for the development of multiple antigenic complex vaccine for toxoplasmosis.Methods: 1. Three different immuning dose 3 109CFU, 3 108 CFU and 3X 107CFU of L.lactis /P30 were administered orally to BALB/C mice. Control group was administered orally with normal saline . After 7 interval within 4 weeks , sera from all animals were tested by ELISA for the presence of total antibodies IgG , and then each group mice were challenged with 100 tachyzoites of the RH strain of T. gondii. The optical immuning dose was determined by comparisonbetween negative control group and immunized groups in the level of specific antibodies and the efficency of immunoprotection. Under the selected immuning dose, L.lactis-P30 was administered orally to BALB/C mice. Control groups were administered orally with gp42 Lactococcus lactis and normal saline respectively. After immunization , sera from all animals were tested by ELISA for the presence of total antibodies IgG and the IgGl and IgG2a antibody isotypes, and then each group mice were challenged with 100 tachyzoites of the lethal RH strain of T. gondii . Then the proliferation of mice spleen lymphocytes were detected by MTT assay and the number of CD4+ and CD8+T cell were achieved by flow cytometer assay . Sera were obtained at different immune time, and were tested for the kinetics of development of antibody IgG. After initial immunization, mice were bled at three-week intervals and sera from all animals were collected. Contents of IFN- Y and IL-2 were measured by ELISA, the NO was detected by enzyme assay.2. The total DNA of Toxoplasma gondii of RH strain was isolated from purified tachyzoites that were obtained by passage through a colum of CF-11 cellulose. The gene fragment encoding ROP2 was amplified by polymerase chain reaction(PCR) from the total DNA.The gene of ROP2 was subcloned into the plasmid pUC119. After the identification by PCR and restriction enzyme cleavage,DNA sequence was determined. The Sac I /Hindin digested fragment of pUC119-ROP2 was inserted into the same site of expression vector pET22b. The recombinant plasmid of pET22b-ROP2 was transformed to a bacterium BL21-Codon Plus(DE3)-RIL and was expressed under the induction of IPTG. The gene fragment encoding P30 was amplified by PCR, and then was subcloned into the recombinant plasmid pUC119-ROP2 that had been constructed. The recombinant plasmid pUC119-ROP2-P30 was digested by Sac I /Hindin and then was inserted into the same site of expression vector pET28b. The recombinant plasmid of pET28b-ROP2-P30 was transformed to a bacterium RIL and was expressed underthe induction of IPTG. Cells were lysed by multiple rounds of sonication. The expression products were analyzed by using SDS-PAGE. The... |
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