Effect Of Adjuvant IL-15 In Parodontopathy DNA Vaccine PVAX1-HA2-FimA/IL-15 On MLN B Cell Proliferation

Objective: Observing the effect of IL-15 as a vaccine adjuvant for parodontopathy DNA vaccine on the proliferation of mesenteric lymph node B lymphocytes in SD rats,and preliminary exploring the mechanism of DNA vaccine for parodontopathy.Methods: The study contains two parts.Part 1 : Identification and preparation of recombinant plasmid pVAX1-HA2-FimA ?pVAX1-HA2-FimA/IL-15.Part 2:Experimental study on the effect of adjuvant IL-15 on B cell proliferation in parodontopathy DNA vaccine.1.Animal selection and grouping: 4-6 week-old SD male rats(n=18),randomly divided into three groups: Experimental group(n=6),negative control group(n=6)and normal control group(n=6).2.Immunization form:Bilateral nasal mucosa dripping slowly,dose of 100?g/ time,increased immunization one time per week,3 times in all.3.Sample collection: Three groups were executed three times after immunization.Lymphocyte suspension was prepared from mesenteric lymph nodes in rats.The purified B cells were obtained by using CD45 R magnetic beads separation method.The purity of B cells was detected by flow cytometry and the activity of cells was determined by trypan blue staining.4.B cell proliferation assay: The obtained B cells were incubated with recombinant FimA protein and HA2 protein for 48 hours,the proliferation of B cells was tested using CCK8 assay;qRT-PCR was used to detect the relative expression of CKs2 mRNA in B cells.ELISA assay detected the expression levels of specific IgA in cell culture supernatant.Results: 1.The parodontopathy DNA vaccines pVAX1-HA2-FimA/IL-15 and pVAX1-HA2-FimA were identified by PCR,restriction enzymes digestion and sequencing.Compared with the NCBI database,the sequence homology was 100%,and no gene mutations were found.2.Mesenteric lymph node B cells were isolated by CD45 R magnetic beads sorting from,in which a very high purity of the sorted population can be obtained using FACS.3.By CCK8 assay,the proliferation of pVAX1-HA2-FimA/IL-15 nasal group was higher than that of pVAX1-HA2-FimA nasal drop group and saline nasal drop group,and the difference was statistically significant(p<0.05);the proliferation of p VAX1-HA2-FimA nasal drops group was higher than saline nasal drops group,the difference was statistically significant(p<0.05).4.The relative expression of CKs2 mRNA in mesenteric lymph node B cells of pVAX1-HA2-FimA/IL-15 nasal group and pVAX1-HA2-FimA group was significantly higher than that of blank control group by qRT-PCR assay,but the difference was not statistically significant(p>0.05).The relative expression of CKs2 mRNA in the lymphocytes of the p VAX1-HA2-FimA/IL-15 group was higher than that in the pVAX1-HA2-FimA group.The difference was not statistically significant(p>0.05).5.Detection by ELISA of secretion of specific IgA levels from mesenteric lymph node B lymphocyte in pVAX1-HA2-FimA / IL-15 nasal drops immunized group-higher than that in saline nasal drops,and in pVAX1-HA2-Fim A nasal dropping group,and the difference was statistically significant(p<0.05);The level of secretory IgA in B lymphocytes of mesenteric lymph nodes in pVAX1-HA2-FimA nasal drop group-SD rats was higher than that in saline nasal drops group,and the difference was statistically significant Significance(p<0.05).Conclusion: IL-15,a vaccine for parodontopathy gene vaccine,as an effective genetic vaccine adjuvant that can promote B cell proliferation and increase the expression level of specific antibody IgA.