Preliminary Study On Immunomodulatory Function Of EspB Encoded By Rv3881c Of Mycobacterium Tuberculosis In Vivo

Objective:To construct recombinant plasmids pGEX-4T-1-EspB and pGEX-4T-1-EspBN.They can stably express the fusion protein of EspB and EspBN.BALB/c mice were immunized with the two proteins to evaluate their immunogenicity.In order to provide experimental basis for screening candidate antigens of tuberculosis vaccine and drug development.Methods:1.The genomic DNA of EspB and EspBN were amplified by PCR and cloned into prokaryotic expression vector.The fusion proteins were induced by IPTG in E.coli BL21(DE3)strains,purified by GST labeled protein purification kit and excised the GST labeled proteins to obtain the target proteins.2.BALB/c mice were immunized with two kinds of protein mixed with equal volume of Freund’s incomplete adjuvant respectively.The same dose of protein and Freund’s incomplete adjuvant were used to enhance the immunity on the 14th and 28th day after the first immunization.The mice in control group were only injected with 100μL PBS solution and equal volume of Freund’s incomplete adjuvant.3.On the 14th,28th and 42th day after the last immunization,the eyeball blood was collected to prepare the antiserum.4.The specificity of each antiserum was detected by Western Blot,and the titer and IgG subtype of each antiserum were detected by ELISA.5.The proliferation of splenic lymphocytes was detected by CCK8 method,the subsets of CD4+ and CD8+was analyzed by flow cytometry,the levels of IL-4 and INF-y were detected by ELISA.Results:1.The recombinant plasmids pGEX-4T-1-EspB and pGEX-4T-1-EspBN were successfully constructed,induced by IPTG and stably expressed fusion proteins in E.coli.2.After immunizing mice with EspB and EspBN,the antisera obtained were specific.3.The titers of total IgG and IgG subtypes in anti EspB protein serum and anti EspBN serum were higher than those in control group.On the first 28 days after the last immunization,the total IgG of anti EspB protein serum was higher than that of anti EspBN serum(P<0.05).The serum IgG2a titer of anti EspB protein was lower than that of IgG 1 on the 14th day of the last immunization,higher than that of IgG1 on the 28th day of the last immunization,and close to that on the 42th day of the last immunization.4.After 28 days of the last immunization,the proliferation stimulating index of splenic lymphocytes in EspB group was higher than that in control group(P<0.05).On the 42nd day of the last immunization,the stimulation index of spleen lymphocyte proliferation in EspBN group was higher than that in PBS group(P<0.05).5.The results showed that the percentage of CD4+ in spleen lymphocytes in EspB group was higher than that in PBS group 14 days after the last immunization,and that of spleen lymphocytes in EspB group was higher than that in PBS group(P<0.05).6.The release of IL-4 in both EspB group and EspBN group showed a decreasing trend,which was significantly higher than that in PBS group on the 14th day of the last immunization(P<0.05),and significantly lower than that in PBS group on the 42nd day of the last immunization(P<0.05).The release level of INF-γ in EspB group and EspBN group showed an increasing trend,which was significantly higher than that in PBS group 42 days after the last immunization(P<0.05).Conclusion:The results showed that both Mycobacterium tuberculosis EspB protein and EspBN protein could induce humoral and cellular immune responses in varying degrees.