Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene ...Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.展开更多
Objective To construct the eukaryotic expression recombinant plasmid, pcIFN γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3 rhoptry protein 1 (pc ROP1) combined with pcIFN γ aga...Objective To construct the eukaryotic expression recombinant plasmid, pcIFN γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3 rhoptry protein 1 (pc ROP1) combined with pcIFN γ against Toxoplasma gondii (T gondii) infection in mice Methods A fragment of the IFN γ gene was directly inserted into the pcDNA3 plasmid and identified by two restriction endonucleases digestion pcIFN and pcROP1 DNA was injected into the left leg muscle of mice at a dosage of 100?μg, and a booster vaccination was given at the same dosage after two weeks Control groups were injected with pcDNA3 blank plasmid or normal saline At 30, 50 and 70 days after booster injection, kinetic tests were carried out: MTT assay for the proliferation response of T lymphocyte cells and the activity of NK cells, sandwich ABC ELISA for the determination of IFN γ, IL 2 and IL 10; a serum enzymetic aassay for nitric oxide (NO) in sera and ELISA for the titer of IgG antibody in sera Results The recombinant plasmid, pcIFN γ was constructed The proliferation response of spleen T lymph cells, NK cell killing activity, and serum levels of IFN γ, IL 2 and NO in mice injected with pcROP1 and pcIFN γ were higher than in those injected with pcROP1 alone There was no difference in IgG antibody levels between the two groups Conclusion The genetic adjuvant, pcIFN γ, could enhance the cellular immune response induced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection展开更多
文摘Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.
文摘Objective To construct the eukaryotic expression recombinant plasmid, pcIFN γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3 rhoptry protein 1 (pc ROP1) combined with pcIFN γ against Toxoplasma gondii (T gondii) infection in mice Methods A fragment of the IFN γ gene was directly inserted into the pcDNA3 plasmid and identified by two restriction endonucleases digestion pcIFN and pcROP1 DNA was injected into the left leg muscle of mice at a dosage of 100?μg, and a booster vaccination was given at the same dosage after two weeks Control groups were injected with pcDNA3 blank plasmid or normal saline At 30, 50 and 70 days after booster injection, kinetic tests were carried out: MTT assay for the proliferation response of T lymphocyte cells and the activity of NK cells, sandwich ABC ELISA for the determination of IFN γ, IL 2 and IL 10; a serum enzymetic aassay for nitric oxide (NO) in sera and ELISA for the titer of IgG antibody in sera Results The recombinant plasmid, pcIFN γ was constructed The proliferation response of spleen T lymph cells, NK cell killing activity, and serum levels of IFN γ, IL 2 and NO in mice injected with pcROP1 and pcIFN γ were higher than in those injected with pcROP1 alone There was no difference in IgG antibody levels between the two groups Conclusion The genetic adjuvant, pcIFN γ, could enhance the cellular immune response induced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection
