The Molecular Mechanism Of E. Coli Heat-labile Enterotoxin B Subunit (LTB) As Adjuvant

LTB is a powerful mucosal adjuvant, can induce strong systemic and mucosal immune response. LTB increase the response to antigen, but also change the immune reaction type by the binding to GM1. The potential mechanism of LTB immunomodulation activity involves its effect on the differentiation and survival of leukocytes. Up to date, LTB have been found to(i) increase the expression of Th1 and Th2 cytokines;(ii) induce the apoptosis of CD8+ T cells and proliferation of CD4+ T cells;(iii) increase the expression of MHCⅡ, ICAM-1, CD25 and CD40, which could activate B cells;(iiii) affect antigen processing and presentation by macrophages, B cells, However, the mechanism is unknown.Objective: Explore the mechanism of LTB as adjuvant by capture the proteins interacting with LTB; Map the interaction between these proteins; Detect the interaction with immunofluorescence assay.Method:(1) induce His-LTB fusion protein.(2) purify and renature LTB fusion protein.(3) Use Toxin Eraser Kit to remove the toxin from purified LTB fusion protein.(4) Extract the RAW 264.7 cell(treated with LTB 12 h) protein with Protein Interaction Kit and test the protein with SDS-PAGE., identify the pull-down proteins by MS.(5) Predict the possible interacting proteins by bioinformatics software, analyze the function and pathway of these proteins, outline the interaction map.(6) Test the interaction by immunofluorescence assay.(7) Detect the expression of β-actin and GSTO1 by WB.Result:(1) Captured the LTB interaction protein by pull down and identified these proteins by MS.(2) 25 proteins were identified, included Junction plakoglobin,heat shock protein 1, carbamoyl-phosphate synthetase 1, vimentin and so on.(3) Immunofluorescence assay indicated that Vim did not bind with LTB and GM130 interacted with LTB in vivo.(4) After treated with LTB, the RAW264.7 cell expressed β-actin increasedly, the expression of Hspd1 unchanged.Conclusion:From MS and immunofluorescence assay results, After LTB binds to GM1, it was internalized by macrophage with the help of cytoskeletal proteins,tansported to Golgi apparatus by vesicle,and processed in Golgi apparatus. after that it will be tansported to T cell and Jup, Jup activates TCF/LEF to promote the secretion of immunogloblins and cytokines, activate T cells, and induce T cells proliferation and differentiation which contribute to LTB as adjuvant.