| Objective : In our previous study of periodontitis gene vaccine pVAX1-HA2-FimA,pVAX1-HA2-FimA/IL-15,to establish experimental periodontitis model,using two kinds of gene vaccine by nasal mucosal immunization of SD rats,detection of salivary specific SIg A antibody levels,and observation the protective effect of gene vaccine on periodontium.Methods: The article included three parts Part 1 : Extraction and identification of gene vaccine pVAX1-HA2-FimA/IL-15 and pVAX1-HA2-FimA.1、Verification of colony PCR.3、Identification by DNA sequencing.3、The gene vaccine of pVAX1-HA2-FimA/IL-15、pVAX1-HA2-FimA and pVAX1 were extracted in large quantity with endo-free Plasmid Kit.Part 2: Establishment of SD rat periodontitis model1、4-6-week-old SD male rats(n=14)were randomly divided into two groups each group has seven rats.The second molar of the upper jaw in the periodontitis group was ligated with silk thread.2、Gum inflammation the degree of tooth loosening were observed at first,fourth and eighth weeks.The absorption of alveolar bone was observed under X-ray,The length of 6 points in the middle,mesial and distal was measured under the stereo scopic microscope.The average value of the 6 point length was the total alveolar b one absorption.3、The relative expression of TNF-α and MMP-8 m RNA in the gingival tissues of two groups of maxillary second molar teeth was detected by q RT-PCR.Part 3:Experimental Study on periodontitis Gene Vaccine Immunized by nasal mucosa in SD rats.1、Animal selection and grouping: SD male rats(n=54)were randomly divided into nine groups,each group has six rats;group A、B were used as control groups and group C were used as Inflammatory control groups.Group D: 50 μg pVAX1-HA2-FimA group,group E: 100 μg pVAX1-HA2-FimA group,group F: 150 μg pVAX1-HA2-FimA group,group G: 50 μg pVAX1-HA2-FimA/IL-15 group,group H: group 100 μg pVAX1-HA2-FimA/IL-15,group I: Group 150 μg pVAX1-HA2-FimA/IL-15.2、Immunization form:The immunization route was nasal mucosal immunization,all the rats were immunized three times,after the first time immunity,these rats need interval one weeks to strengthen the immunity.3、1 day after the last immunization,the upper second molars of the SD rats were ligated in the inflammatory control group and the experimental group.4、Saliva sample collection: collected saliva samples of SD rats 1 day before the first immunization and on the 7th day after each immunization(until the 7th week).5、Detection of specific antibodies: indirect ELISA method was conducted to detect SIg A in saliva.6 、 The relative expression of TNF-α and MMP-8 m RNA in gingival tissue were identified by q RT-PCR,the measurement of alveolar bone absorption,and safety testing in all of groups.Results:1、PVAX1-HA2-FimA and pVAX1-HA2-FimA/IL-15 were identified by PCR and DNA sequencing,compared with NCBI database,sequence homology 100%,no gene mutation.2、In periodontitis group,gingivitis gradually increased,tooth mobility increased,alveolar bone absorption increased.The expression of TNF-α and MMP-8 m RNA of the gingival tissues were increased in,the differences was statistically significant(P < 0.01).3、The weight of SD rats in each experimental group showed a gradual upward trend,and there was no significant differences between the groups at the same time point.4、Immunized SD rats with gene vaccines,anti-FimA antibody of SIg A began to rise at 1st week,continued to increase gradually between 2nd-4th week and reached its peak at4 th week,antibody level declined slowly from 5th week,lasting 8 weeks.The level of antibody in pVAX1-HA2-FimA/IL-15 group with the same dose of vaccine 3-7 week was higher than that in pVAX1-HA2-FimA group.Specific antibodies level of anti-HA2 antibody of SIg A began to rise at 1st week,continued to increase gradually between 2nd-4th week and reached its peak at 4th week,antibody level declined slowly from 5th week,lasting 8 weeks.The level of antibody in pVAX1-HA2-FimA/IL-15 group with the same dose of vaccine 3-7 week was higher than that in group pVAX1-HA2-FimA.5、The relative expression of TNF-α and MMP-8 m RNA of gingival tissues in each group was higher than that in the control group,and was lower than that in the inflammatory control group,and the differences was statistically significant(P < 0.05).6、The alveolar bone absorption of the pVAX1-HA2-FimA/IL-15 group with the same dose of the vaccine was compared with the pVAX1-HA2-FimA group was less,and the differences was statistically significant(P < 0.05).7、No significant abnormal pathological changes were found in the pathological sections of the main organs of SD rats in the experimental group and the control groupConclusion:Gene vaccine pVAX1-HA2-FimA/IL-15 and pVAX1-HA2-FimA can successfully triggered the immune response by nasal mucosal immunization,which has immunogenicity.Specific antibodies are produced in the induced saliva and can protect periodontal tissues by gene vaccine.Using of immunoadjuvant IL-15 can improve the antibody expression level and had significantly better protective effect on periodontal tissue. |
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