Differences In The Specific Expression Levels Of IL15 In Tuberculosis Peripheral Blood And The Efficacy Of DNA Vaccine With IL7

Objective(1)Specific expression levels of IL15 can be studied in peripheral blood samples of tuberculosis patients,especially in the latent tuberculosis group.(2)DNA vaccine p VAX1-IL7-Ag85A was constructed and its immune protective effect was tested.To explore the immune response performance of the vaccine in mouse immune experiments and evaluate the role and potential of the vaccine in preventing TB infection.Methods(1)According to the clinical sample sources,total 100 samples were collected and divided into four groups:the active tuberculosis group,the latent tuberculosis group,non-tuberculosis group(other respiratory disease)and the healthy control group.And then the peripheral blood was sampled respectively.Each blood sample was stimulated with identified mycobacteria latent related antigen Rv2626c,Rv2031,Rv2028c,Rv2029c,Rv3406 and Rv1738 respectively,the concentration of IL15 was determined by ELISA after 48 hours.(2)Obtain the fragment of IL7 and Ag85A by PCR,double digestion technique then connection and transformation were used to insert the two fragments into expression plasmid p VAX1-IL7-Ag85A.Picking the positive clones for the further identification.Toal 19 femal specific-pathogen free(SPF),5-6 weeks old Balb/c mice were devided into four groups randomly,4 in PBS control group,5 in p VAX1,5 in p VAX1-IL7 group5 and in the p VAX1-IL7-Ag85A group,to assay the immunogenicity of p VAX1-IL7-Ag85A.Another total 19 same femal SPF mice were obtained and divieded into 4 groups evenly as abovely,5 mice of each group.Mice were immunized once every 2weeks and lasted for 6 weeks,then 19 mice were killed at 2 weeks later,the other 19 mice were challenged with Mtb H37Rv 3 weeks after finished immunization.The lung and spleen lymphocyte were sampled for flow cytometry analysis and bacterial CFU(colony forming units)assay.Results(1)The expression level of IL15 was changed in the absence of latent infection associated antigen.The expression level of IL15 in latent group was significantly higher than that of active TB group,non-tuberculosis disease and healthy control group(p<0.001).The difference was most pronounced after Rv2031 antigen stimulation.(2)This construct of p VAX1-IL7-Ag85A was sequenced and identified in this experiment.The p VAX1-IL7-Ag85A can induce more significant multifunctional CD4~+T cells IFN-γ~+TNF-α~+subgroup immune response.The continuous mice immunized with p VAX1-IL7-Ag85A and then challenged with Mtb H37Rv revealed its better immune protective effects of this construct,especially the lung CFU counting and HE staining.Conclusion(1)The expression level of IL15 has been changed,indicating that IL15 itself may be associated with immune protection.After latent related antigen stimulation,latent TB group obviously higher than that of ATB,non-tuberculosis other respiratory disease and healthy control group,especially in Rv2031 stimulated group,the difference is most obvious,with potential as an alternative antigen to diagnose active tuberculosis and latent tuberculosis infection with IL15 expression level.(2)This experiment successfully established and indentified the constructof p VAX1-IL7-Ag85A,animal infection experiment exhibited that p VAX1-IL7-Ag85A has a certain induction effect on multifunctional CD4~+T cells in mice,especially IFN-γ~+TNF-α~+T cells immune response and CD8~+memory T cell responses.