Animal Experiment Study On The Changes Of PIgR And SIgA Content In Mice Induced By Different Doses Of Anti-caries DNA

Objective:The mice were immunized with different doses of anti-caries DNA vaccine through the nasal mucosa,and the dynamic changes of pIgR and SIgA under the action of the anti-caries DNA vaccine were detected.So as to reveal the appropriate immunization dose of the anti-caries DNA vaccine for mice,which laid a theoretical foundation for the application of DNA vaccine.Methods:1.Identification and extraction of recombinant plasmid pVAX1-SA:The Escherichia coli containing the recombinant plasmid pVAX1-SA is resuscited,and the bacterial liquid PCR and enzyme digestion are used to identify whether the recombinant plasmid pVAX1-SA is correct.The plasmid is extracted according to the instructions of Tiangen endotoxin-free plasmid kit.2.To immunize BALB/c mice with anti-caries DNA vaccine:(1)Animals and groups: Healthy BALB/c mice aged 6-8 weeks,male or female,weighing 18-22 g.The mice are randomly divided into three groups: group A(28 pVAX1 control group),group B(28 in the negative control group),group C(84 in the experimental group).(2)Immunization program: group A was instilled with empty vector pVAX1 through nasal mucosa;group B was instilled with sterile normal saline through nasal mucosa;group C was randomly divided into three groups of 50?g,100?g,and 200?g according to the immunization dose.,The mice were immunized by dripping through nasal mucosa for 3 times.,the first immunization was followed by the second immunization at an interval of 1 week,and the second immunization was strengthened at an interval of 1 week again.(3)Sample collection and detection: a.At 24 h,48h,and 96 h after the second immunization,6 mice in each group were randomly sacrificed,and the small intestine and submandibular gland were taken.q RT-PCR analysis of the relative expression of pIgR and Spap/A in the small intestine and submandibular gland;b.Collect saliva and blood before immunization and 2 weeks,4 weeks,and 8 weeks after the first immunization,and detect SIgA and IgG antibodies,IL-10 and pIgR levels by ELISA;c.Immunohistochemical localization and quantitative expression of pIgR and IL-10 in tissues;d.HE undergoes pathological examination.Results: 1.Bacterial liquid PCR and KpnI,EcoR I double enzyme digestion to identify pVAX1-SA,electrophoresis results in the 1.3kb site of the amplified fragment and the size of the target fragment Spap/A.2.The expression of pIgR and target antigen Spap/A in submandibular gland and small intestine of mice were detected by q RT-PCR.The results showed that the expression of pIgR and Spap/A in the experimental group relative to the control group showed an upward trend within 96 hours after the second immunization,and the expression of pIgR and Spap/A in each tissue of the 100 ?g group was higher than that in the 50 ?g and 200 ?g groups(p<0.05).3.ELISA was used to detect the levels of pIgR,SIgA in mouse saliva and IgG in serum.The results showed that the antibody level of the control group did not change significantly from 2 to 8 weeks,and the SIgA and IgG of the experimental group increased gradually,and the antibody level of the 100?g group was the highest(p < 0.05).The pIgR level in the saliva of the experimental group was lower than that in the control group.At the 4th week,the pIgR level in the saliva of the 100?g group was the lowest compared with each group,which was statistically significant(p<0.05).4.Immunohistochemical localization and quantification of pIgR expression in tissues.In the small intestine,pIgR is mainly located on the surface of intestinal epithelial cells,mucus cells and intestinal villi.In the submandibular gland,pIgR is mainly expressed around acinus.The expression level of pIgR in small intestine and submandibular gland tissue of experimental group was higher than that of control group(P <0.05).5.The levels of IL-10 in saliva and serum in 200?g group and 100?g group were detected by ELISA.The results showed that the level of IL-10 in saliva and serum was significantly increased in the 200?g group compared with the 100?g group(p<0.05).Immunohistochemistry was used to locate and quantify the expression of IL-10 in the spleen,submandibular gland,and small intestine tissues.The expression level of IL-10 in each tissue in the 200?g group was higher than that in the 100?g group The spleen expression level was the highest and the small intestine was the lowest.Conclusion:Different immunization doses of anti-caries DNA vaccines were instilled into mice through nasal mucosa.At the level of transcription and protein,100?g was better than 50?g and 200?g groups.100?g was more effective in inducing the body to produce an immune response and stimulate the body to express pIgR.And participate in the high-efficiency transport of SIgA,,which is the best in terms of immune and economic effect.For this experiment,the appropriate immune dose for the nasal mucosal infusion immune pathway is obtained.