Construction Of Recombinant Plasmid Containing Toxoplasma Gondii HSP70 Gene And Cellular Immunologic Response Of Mice Induce By Recombinant Plasmid

ObjectiveThe present study is focused on the construct of p3×Flag-CMW-14-TgHSP70 from Toxoplasma gondii RH strain and the cellular immunologic response of BALB/c mice induced by subdermal immunization with recombinant plasmid p3×Flag-CMW-14-TgHSP70 at different doses.MethodsThe first part: The primers of HSP70 gene were designed and the HSP70 gene was amplified by PCR. Gene fragment of Toxoplasma gondii heat shock protein (TgHSP70) were obtained. Gene fragment were double enzyme digest, then connected with eukaryotic expression plasmid p3×Flag-CMW-14, connection products were identification by enzyme digestion, PCR and DNA sequencing. The recombinants were transfected to HEK 293T cells by Entranster TM-D. TgHSP70 expression products were identified by both gene and protein levels of RT-PCR and Western blot.The second part: 125 BALB/c mice were randomly divided into 5 groups. The mice were immunized with 50μg, 100μg, 150μg p3×Flag-CMW-14-TgHSP70 by subcutaneous injection, per mouse 3 times at an internal 2 weeks, control group was injected with 100μl elution buffer, empty vector group were injected with 50μg p3×Flag-CMW-14. Recombinant plasmid and empty plasmid were dissolved in 100μl elution buffer. The murine sera were separated at week 2, 4, 6. 8 and 10 after the first immunization, levels of IL-2 and IFN-γin sera were measured by ELISA. Spleen lymphocytes were sterility separated and counted, and then, adding a final concentration of 5μg/ml of ConA, 5%CO₂ incubator at 37℃to promote lymphocyte transformation about 24 hours, collecting cells supernatant at 24h, 72h respectively. Levels of IL-4 and IL-2 in supernatant of 24h cultured spleen lymphocytes and IFN-γlevels of supernatant of 72h cultured spleen lymphocytes were measured by ELISA. ResultsA total of 495 HSP70 fragment genes were amplified by PCR. Construction of recombinant plasmid p3×Flag-CMW-14-TgHSP70 was successful. Positive clone were identification by enzyme digestion, PCR and DNA sequencing, gene sequence were completely correct. The p3×Flag-CMW-14-TgHSP70 eukaryotic expression plasmids were transfected into HEK 293T cells. The positive band of expected size were displayed by RT-PCR, HSP70 gene's express products were approximately 19 kDa which displayed by western-blot analysis.The levels of serum IL-2 in 50μg, 100μg, 150μg recombinant plasmid groups increased at 6 week, reached peak at 8 week and slightly down at 10 week after first immunization. Although the levels of serum IFN-γin 50μg, 100μg, 150μg recombinant plasmid and control groups were consistent always, no significant difference. Spleen cells in 50μg (17.362×10⁶个/ml) recombinant plasmid group was significantly higher than 150μg (15.22×10⁶个/ml), 100μg (15.66×10⁶个/ml) recombinant plasmid group, empty plasmid group (14.82×10⁶个/ml) and control group (16.076×10⁶个/ml) at 10 week after first immunization (F=4.478, P<0.05). The level of the IL-4 in spleen lymphocytes in 150μg recombinant plasmid group was significantly higher than 50μg and 100μg recombinant plasmid group from 2 to 10 week, but the difference was not statistically significant. The levels of the IL-2 in spleen lymphocytes 50μg, 100μg, 150μg recombinant plasmid group and the empty plasmid group were higher than control group from 2 to 10 week, the difference was significant (F=5.319, P<0.05). The level of the IFN-γin spleen lymphocytes 150μg recombinant plasmid group was significantly higher than other groups from 2 to 10 week (F=3.918,P<0.05), and reached the highest level at 8 week after first immunization (A₄₀₅=0.9867).ConclusionRecombinant eukaryotic expression plasmid of HSP70 has been successfully constructed. Recombinant p3×Flag-CMW-14-TgHSP70 of different doses induced cellular immune response in BALB/c mice. The levels of the IL-2 and IFN-γin spleen lymphocytes in 150μg recombinant plasmid group were heighter than 100μg, 50μg recombinant plasmid group. Cellular immune response of 150μg, 100μg, 50μg recombinant plasmid group were reached peak at week 8 after immunization. Antigen molecules TgHSP70 which expressed by recombinant plasmid has immunogenicity and could have the potential to be developed as a candidate antigen for toxoplasmosis vaccine.