Research On The Adjuvant Activition And Mechanism Of E.coli Heat-labile Enterotoxin B Subunit (LTB) Mutants

Escherichia coli heat-labile enterotoxin B subunit(LTB)is a kind of mucosal immune adjuvant,the research has found that LTB can combine with cell surface ganglioside(GM1),and play a role in that.In addition,other related signaling mechanism still is not clear.Therefore,the mechanism of LTB adjuvant still is bottleneck for pushing further research.As so far,mucosal adjuvant is not applied in clinic.in this research four LTB mutants were constructed and hope to find some improved LTB adjuvants and further to study the mechanism of adjuvant of LTB.Objective: This thesis researched the adjuvant activity of wild LTB and its mutants,preliminarily evaluate the effect of LTB mutants on activity of mucosal immune adjuvant.To find the strong LTB mutants and analyze the mechanism of wild LTB and its mutants.Methods: Pfu DNA Polymerase was used to point mutate LTB,and constructed recombinant plasmids.The positive clones were confirmed by PCR,endoenzyme assay and DNA sequencing.The target proteins were detected by SDS-PAGE and purified with Magnetic Nickel Beads,and concentrated with PEG8000.The purified LTB,LTB26,LTB34,LTB57 and LTB85 proteins were removed endotoxin and administrated Balb/c female mice(mice vital signs is normal during immune)joint VP8 respectively.The serum and lung lotion were collected and the Ig G and s Ig A levels in serum levels and in bronchi alveolar fluids were detected by ELISA.The NLRP3,AKT1,MAP2K3 and MAP2K4 in nasal mucosal tissue and spleen tissues of immune mice were detected with immunohistochemical assay.Results: 1.p ET32a-LTB26,p ET32a-LTB34,p ET32a-LTB57 and p ET32a-LTB85 mutants were successfully constructed.2.The fusion His-tag-LTB26,His-tag-LTB34,His-tag-LTB57 and His-tag-LTB85 proteins were expressed in E.coli BL21(DE3).ELISA results showed that mutants LTB26 and LTB34 have adjuvant activity,and Ig G and s Ig A levels were higher than that of LTB obviously.Preliminary screening for LTB26 and LTB34 mutants,and its adjuvants were higher than LTB.3.Immunohistochemil indicated that expressed level of NLRP3 is decreased in nasal in groups of LTB+VP8,LTB26 +VP8 and LTB34+VP8,and was not expressed in spleen in all groups.Similarly,NLRP3 in spleen tissue in groups of LTB+VP8,LTB26 +VP8 and LTB34+VP8 showed a similar trend.The expression of AKT1 in group LTB26+VP8 was higher than VP8 group,and in other groups it was lower than VP8 group,in spleen in groups of LTB+VP8,LTB26+VP8 and LTB34+VP8,where it expressed in higher level in groups of VP8 and LTB57+VP8(p<0.05).The expression of MAP2K3 was lower in nasal tissue in groups of LTB+VP8,LTB26+VP8 and LTB34+VP8 than VP8 and LTB57+VP8 groups(p<0.05),and its expression was higher in spleen LTB+VP8,LTB26+VP8 and LTB34+VP8 groups than in groups of VP8 and LTB57+VP8(p<0.05).The expression of MAP2K4 was higher in nasal in groups of LTB+VP8 and LTB26 +VP8,except group of LTB34+VP8,and it was higher in groups of LTB+VP8,LTB26 +VP8 and LTB34+VP8 in spleen.Conclusion:1.This thesis successfully expressed His-tag proteins,His-LTB26,His-LTB34,His-LTB57 and His-LTB85.2.Adjuvant activity of LTB26 and LTB34 had been improved compared with LTB.3.Preliminary speculated NLRP3 was no contribution to the LTB and LTB26 mutant adjuvant activities.Both the AKT1 and MAP2K4 were associated with the adjuvant activities of LTB and its mutants(LTB26,LTB34)in spleen.However,AKT1 might be associated with the adjuvant activity of LTB26 mutant.MAP2K4 was associated with the adjuvant activities of LTB and LTB26,MAP2K3 was associated with the adjuvant activities of LTB and its mutants negatively in spleen.However,MAP2K3 was associated with the adjuvant activities of LTB and its mutants positively in nasal.These differences were laid the foundation for the uncover of the mechanisms of LTB adjuvant activity in future.