Construction of Recombinant Plasmid Containing Toxoplasma Gondii HSP70 Gene and Cellular Immunologic Response of Mice Induce by Recombinant Plasmid
|Keywords||Toxoplasma gondii Heat shock protein 70 Clone Recombinant Eukaryotic expression Gene vaccine Cellular immunity|
Objective To construct Toxoplasma gondii RH strain p3 × Flag-CMW-14-TgHSP70 eukaryotic expression recombinant plasmid. Observation on the cellular immune response of different doses of the recombinant plasmid p3 × Flag-CMW-14-TgHSP70 subcutaneous intramuscular immunizing BALB / c mice induced dynamic change. The first part of the method: to design synthetic TgHSP70 primer, PCR amplified gene fragment of Toxoplasma gondii heat shock protein (TgHSP70) gene fragment encoding double digested with eukaryotic expression plasmids p3 × Flag-CMW-14 connection, The ligation products were identified by restriction enzyme digestion, PCR and sequencing. Lipofectamine transfection recombinant HEK 293T cells, and the expression product of TgHSP70 in two levels of the genes and proteins were identified by RT-PCR and Western blot. Part II: 125 BALB / c mice were randomly divided into five groups, respectively 50μg, 100μg, 150μg recombinant plasmid p3 × Flag-CMW-14-TgHSP70 / only subcutaneously intramuscularly immunized mice three times, each 2-week intervals , blank control group was injected with 100μl Elution buffer empty plasmid group injection of 50μg p3 × Flag-CMW-14 recombinant plasmid and empty plasmid were dissolved in 100μl Elution buffer. 2, 4, 6, 8, 10, respectively, in the first free eyeball blood collection, the serum was separated, each group of five ELISA assay serum IL-2, IFN-γ levels. The mice were killed by cervical dislocation, sterile spleen, isolated splenic lymphocyte count; then added to a final concentration of 5μg/ml of ConA, 5% CO2, 37 ° C cultured 24h, 72h, take the culture supernatant. ELISA assay culture 24h supernatant IL-4, IL-2 and cultured 72h in the supernatant IFN-γ levels. HSP70 encoding gene fragment of approximately 495 bp PCR amplification, p3 × Flag-CMW-14-TgHSP70 recombinant successfully constructed entirely correct positive clones were double digestion, PCR and sequencing, gene fragment sequences. P3 × Flag-CMW-14-TgHSP70 eukaryotic expression recombinant plasmid transfected HEK 293T cells, the visible target gene band of the expected size was detected by RT-PCR, Western-blot detection of expression is the product of about 19 kDa. 50 μg, 100μg, 150μg recombinant plasmid serum levels of IL-2 in the six weeks after immunization increased significantly, peaked eight weeks, 10 weeks a slight decline. 50μg, 100μg, 150μg recombinant plasmid group and the control group, serum IFN-γ levels over time to change basically the same, no significant difference. 10 weeks after immunization, 50μg of recombinant plasmid group (17.362 × 10 ~ 6 / ml) the number of spleen cells was significantly higher than 150μg group (15.22 × 10 ~ 6 / ml), 100μg (15.66 × 10 6 / ml ), empty plasmid group (14.82 × 10 ~ 6 Ge / ml) and the control group (16.076 × 10 ~ 6 Ge / ml) (F = 4.478, P lt; 0.05). 2 to 10 weeks after immunization the 150μg group of spleen lymphocytes IL-4 water on average higher than 50μg and 100μg group, but the difference was not statistically significant. 2 to 10 weeks after immunization, 50μg, 100μg, 150μg recombinant plasmid group and empty plasmid group spleen cells from IL-2 levels were higher than that in the control group, the difference was significant (F = 5.319, P lt; 0.05), 150μg recombinant plasmid group significantly higher than the other groups. 2 to 10 weeks after immunization, 150μg recombinant plasmid spleen cell IFN-γ levels were significantly higher than the other groups (F = 3.918, P lt; 0.05), and in the first eight weeks (A405 = 0.9867), the highest value. Conclusion We successfully constructed the eukaryotic expression recombinant plasmid p3 × Flag-CMW-14-TgHSP70. Different doses of recombinant plasmid p3 × Flag-CMW-14-TgHSP70 immunized mice could induce a cellular immune response, 150μg recombinant plasmid group induced the spleen lymphocyte IL-2 and IFN-γ levels than 50μg, 100μg recombinant plasmid group 50μg, 100μg, 150μg recombinant plasmid group 8 weeks after immunization induced cellular immune responses peaked. The recombinant plasmid expression the antigen molecule TgHSP70 immunogenic, and can be used as a vaccine candidate antigens in-depth study.