The Effects Of Transgenic Tomato Anti-Caries Vaccine On The Number Of T And B Lymphocytes In BALB/c Mice

Objective:By detecting the percentage of T and B lymphocyte subsets out of total number of T and B lymphocytes in the spleen of BALB/c mouse after oral immunization with transgenic tomato anti-caries vaccine,the difference in the number of T and B lymphocyte subsets among the groups was analyzed to preliminarily explore the mechanism of action of the transgenic tomato anti-caries vaccine.Method:1.The PAcA protein antigen and rabbit polyclonal antibody were prepared,and the T₂transgenic tomato plants were cultivated.The T₂ transgenic tomato fruits were identified by PCR,Western blot and ELISA,and the expression of fusion protein in transgenic tomato was detected.2.18 male and 6-week-old BALB/c mice were divided into three groups according to the principle of random grouping:blank group?normal feeding?,negative control group?normal tomato juice?,experimental group?T₂ transgenic tomato juice?,6 mice in each group.After 4 weeks of intragastric immunization?once per week?,the antibody titers of SIgA in saliva and IgG in serum were detected by ELISA;the spleens of each group were taken at 5th week after the first immunization and be made into single cell suspension.Flow cytometry was used to detect the percentage of T and B lymphocyte subsets out of total number of T and B lymphocytes in the spleens of each group,and the difference of subpopulations among the groups was analyzed.Results:1.The PAcA protein was purified by affinity chromatography and the concentration of purified PAcA protein was determined to be 2.19 mg/mL.2.The T₂ transgenic tomato plants were confirmed to contain the pacA gene after being identified by PCR.The results of Western blot showed that the T₂ tomato fruit expressed the target protein,indicating that the T₂ transgenic tomato was successfully cultured,and the concentration of fusion protein in transgenic tomato was determined to be 7.929?g/mL.3.After immunization of BALB/c mice with transgenic tomato,SIgA antibody and IgG antibody were respectively detected in saliva and serum,and the antibody titer reached the peak at 5th week after the first immunization.4.At 5th week after the first immunization,the percentage of activated B cells,memory B cells and plasmablasts of the mice's spleens in the experimental group was significantly higher than both blank group and negative control group?P<0.05?;The percentage of CD4⁺T cells of the mice's spleens in the experimental group was significantly higher than both blank group and negative control group?P<0.05?.The percentage of CD8⁺T cell of the mice's spleens in the experimental group was lower than both blank group and negative control group?P>0.05?;the ratio of CD4⁺/CD8⁺of the mice's spleens in the experimental group was significantly higher than both blank group and negative control group?P<0.05?.Conclusion:After oral immunization with transgenic tomato anti-caries vaccine in BALB/c mouse,activated B cells,memory B cells,plasmablasts and CD4⁺T cells can be activated to effectively stimulate the body to produce an immune response.