[Cloning and identification of ROP2 gene of Toxoplasma gondii].

OBJECTIVE: To amplify ROP2 from the genomic DNA of Toxoplasma gondii RH strain and construct eukaryotic expression plasmid pc-DNA3-ROP2. METHODS: Tachyzoites of T. gondii RH strain were collected and depurated to obtain genome. A pair of primers was designed and synthesized according ROP2 gene sequence. The gene fragment encoding ROP2 was amplified from the genomic DNA of T. gondii RH strain by means of PCR. It was then reclaimed and purified, and inserted into cloning vector pUCm-T. The recon was cut by EcoR I, Hind III, and the inserted ROP2 gene fragment was subcloned into pc-DNA3 eukaryotic expression vector using T4DNA ligase, followed by further PCR identification, double digestion via restrictive enzymes, and sequencing. RESULTS: The amplified specific gene fragment of ROP2 was about 1.7 kb in length. The gene fragment cloned and subcloned into pc-DNA3 was correct, and the eukaryotic expression plasmid contained ROP2 gene fragment was successfully constructed. CONCLUSION: The recombinant plasmid pc-DNA3-ROP2 was successfully constructed.

[Studies on the immuno-protection of ROP2 nuclei acid vaccine in Toxoplasma gondii infection].

OBJECTIVE: To study the protective effect of ROP2 nuclei acid vaccine in mice. METHODS: Forty-two BALB/c mice were divided into three groups. Each mouse in experiment group was injected with 50 microg recombinant plasmid pc-DNA3-ROP2 through musculus quadriceps fexoris. In control groups, each mouse was injected with 50 microg blank plasmid pc-DNA3 and with 50 microl PBS respectively. All mice were immunized for three times with an interval of three weeks. The volume was doubled for the final injection in the two plasmid groups. Blood, spleens and lymph nodes of 4 mice in each group were taken for the detection of CD4+, CD8+ T cells and cytokines 2 weeks after the final immunization. The rest mice in 3 groups were challenged with 500 tachyzoites of Toxoplasm gondii RH strain for further observation. RESULTS: The vaccine induced strong cellular and humoral immune response. The titer of antibody in serum was high after inoculation and recognized ROP2 protein antigen expressed in vitro. The lymphocyte phenotype was analyzed. CD4+ T cells proliferated sharply (69.5+/-3.4)%, and the ratio of CD4/CD8+ increased considerably by (4.69+/-1.32)% (P<0.01). The level of IL-2, IL4, IL-6, IL-12, IFN-gamma and TNF in serum and cultured supernatant of spleen cells and lymph cells was higher in the experiment group than that in control groups, especially in serum. 88.9% mice in the experiment group were protected 180 hours after the challenge of T. gondii. The death time of mice in experiment group was delayed and the survival time was prolonged in comparison to that in control groups with a significant difference (P< 0.01). CONCLUSION: The recombinant ROP2 nuclei acid vaccine shows fair immunogeni-city and obviously produces immuno-protection.