The Effect Of Transgenic Tomato Anticaries Vaccine On The Gene Expression Of The SD Rats Spleen

Objective:The PAcA protein and polyclonal antibody was constructed based on the reserved seeds of T₁ transgenic tomato anti-caries vaccine and E.coli containing recombinant plasmid pET30a-PAcA fabricated in the earlier stage.Then T₂ transgenic tomatoes were cultured and SD rats were immunized with pure tomato juice.Then the changes of spleen gene were sequenced by RNA-seq to explore immune mechanism of transgenic tomatoes anti-caries vaccine.Methods:1.The identification of recombinant plasmid pET30a-PAcA by PCR and sequencing technology.2.E.coli was induced by IPTG to express PAcA protein,and then broken by ultrasound.PAcA protein was purified and identified by Western Blot.3.Immunized New Zealand white rabbits with PAcA protein,the polyclonal antibody was obtained from serum.4.The gus and pacA gene in T₂ generation transgenic tomatoes was detected by PCR,tomato fruits contained exogenous gene APB-DOCK8 was selected.5.The total protein concentration was detected by BCA,the fusion protein concentration was detected by ELISA,the expression of fusion protein in the fruits of T₂ transgenic tomatoes was detected by Western Blot.6.Animal experiment:（1）Animal selection and grouping:3-weeks-old healthy female SD rats（n=25）were randomly divided into five groups（five in each group）,and the caries model was established in each group.Group A:immunized by 2 mL/kg transgenic tomato juice;Group B:immunized by 4 mL/kg transgenic tomato juice;Group C:immunized by 6mL/kg transgenic tomato juice;Group D:immunized by 8 mL/kg transgenic tomato juice;Group E:immunized by 10 mL/kg transgenic tomato juice.（2）Immunization time:once a week for four consecutive weeks.（3）Sample collection:Saliva and blood samples were collected 1 day before the first immunization and 7 days after each immunization up to 7weeks.（4）Antibody detection:SIgA specific to PAcA in saliva and IgG specific to PAcA in serum were detected by ELISA.（5）The maxillary and mandibular bones were taken for Keyes dental caries score.The experiment was approved by the Animal Ethics Committee of Zunyi medical university.7.Transcriptome sequencing:The experiment was carried out after the optimal immune concentration of transgenic tomato juice and sampling time was confirmed in animal experiment 1.（1）3-weeks-old healthy female SD rats（n=20）were randomly divided into two groups（10 in each group）.The experimental group was fed with 8 mL/kg of transgenic tomato juice and the other group was fed with 8 mL/kg normal tomato juice.（2）The other steps were the same as animal experiment 1.（3）SD rats were sacrificed at the6th week.Total RNA of spleens was extracted and mRNA was enriched by Oligo dT magnetic beads and used for RNA-seq.The caries were scored as before.The experiment was approved by the Animal Ethics Committee of Zunyi medical university.Results:1.PCR and plasmid sequencing showed that the plasmid of E.coli contained pacA gene.Purified PACA protein concentration was 0.8284 mg/mL.Polyclonal antibody was obtained after New Zealand white rabbits immunized by PACA protein.2.The marker gene gus and the target gene pacA in T₂ transgenic tomato fruit was detected by PCR,then the positive tomato fruit was obtained.3.The total protein concentration of T₂ generation tomato fruit was 14.77 mg/mL and fusion protein concentration was 36.28μg/mL（0.2456%）.Fusion protein of T₂ generation transgenic tomato was 173.5 KD,identified to the APB-DOCK8 molecular size.4.Animal experiments showed that the highest level of SIgA and IgG in group D was at the 6th week,the results was significant different with the control group（P<0.05）.The scores of dental caries in group D were also significantly different from those in the control groups（P<0.05）.5.RNA extracted from SD rats spleen showed high quality,purity and good biological repeatability.RNA-seq sequencing showed that 40 genes with significant difference in expression of mRNAs（P-adjust<0.05&|Fold Change|≥1.5）,26 genes were significantly up-regulated and 14 genes were significantly down-regulated.Up-regulated gene was annotated to 88 items by GO analysis,including humoral immune response,regulation of B cell activation and so on,F<0.001.Down-regulated gene was annotated to307 items by GO analysis,but F>0.4.KEGG analysis showed that Up-regulated gene was annotated to 56 signaling pathways,including Fc gamma R-mediated phagocytosis,intestinal immune network for IgA production and B cell receptor signaling pathway,were up-regulated,F<0.001.Down-regulated gene was annotated to 16 enriched signal pathways,F>0.1.Conclusion:1.PAcA protein and polyclonal antibody was made.Transgenic tomatoes containing marker gene gus,target gene pacA and target protein APB-DOCK8 to prevent caries were cultivated,which provided foundation for animal experiments.2.After the SD rats were immunized with GM tomato juice by gavage,the most antibodies were produced in serum and saliva at the dose of 8 mL/kg at the 6th week.3.Specific antibodies were produced in SD rats immunized with transgenic tomato anti-caries vaccine.RNA-seq technique was used to analyze the spleen genes of SD rats.Among them,26 genes were significantly up-regulated,and 14 genes were significantly down-regulated.GO analysis of up-regulated genes was significantly enriched in functions related to humoral immunity,such as B cell activation,humoral immune response,KEGG analysis was significantly enriched in B lymphocyte activation,antibody production and other signaling pathways;GO analysis of down-regulated genes and KEGG analysis had no significant difference,so it was determined that the vaccine could achieve humoral immune effect by up-regulating spleen-related immune genes for further study.The study of vaccine mechanism provides theoretical basis.