Prokaryotic Expression Of MBP And Its Effects On Phenotypical And Functional Maturation Of Dendritic Cells

Background and ObjectivesMaltose binding protein (MBP) found recently is a kind of immunoadjuvant and it can improve the immunogenicity of recombinant subunit vaccine. Some studies showed that MBP had extensive immune activity which could activate NK cells, macrophages, or Thl cells. Moreover, MBP could induce the maturation of dendritic cell (DC) and the production of proinflammatory cytokines via TLR4. These results indicated that MBP could be used as immunopotentiator which could improve the immune response to panthogens.DC is the most powerful antigen-presenting cells (APC) and the only one which can directly activate naive T cells. It plays an important role in innate and adaptive immunity. DC exist in two functionally and phenotypically distinct states, immature and mature. Immature DC express relatively low level of MHC molecules and costimulatory molecules------CD40, CD54, CD80, CD86, et al, while mature DC express high level of MHC molecules, and costimulatory molecules,such as CD80(B7.1), CD86(B7.2), CD40, and intercellular adhesion molecule (ICAM-1, CD54).Currently, the researches about MBP primarily focus on the activity of its recombinant protein. However, it is still a question about the mechanisms how MBP act on immune system and improve the immune response of vaccines. Therefore, we construct the prokaryotic expression system of MBP and MBP is expressed and purified. Then, we examined the effects of MBP on the phenotypical and functional maturation of DCs to research the molecular mechanism of the new immunopotentiator MBP.Methods1. Using genomic DNA of E.coli K12as the template, PCR was performed to amplify entire malE gene and then constructed the prokaryotic expression systems of malE gene. Expression of the target recombinant protein MBP was detected by SDS-PAGE and the expressed MBP were extracted by Amylose Resin. Then the immunoreactivity of MBP was tested by Western Blot.2. DC2.4cell line culture:DC2.4, a cell line of DC originated from C57BL/6mice, was cultured in complete medium (RPMI-1640containing10%FCS,300mg/L glutamine,100U/ml penicillin,100μg/ml streptomycin).3. There are3groups,1) blank group;2)1μug/ml MBP;3)5μg/ml MBP. DC2.4were collected and treated as follows:1) blank group, i.e. DC2.4(1×106cells in a total volume of1ml per well);2) DC2.4+1μg/ml MBP;3) DC2.4+5μg/ml MBP. The24-well plates were incubated at37℃,5%CO2for10h. Culture supernatants and cells were collected for measurement of cytokines and flow cytometry analyses, respectively.4. Phenotypic analyses by flow cytometry:Expression of CD40, CD54, CD80, CD86, MHC-I and MHC-II on the surface of DCs were detected by flow cytometry.5. ELISA:the concentrations of IL-1β, IL-6, IL-12and IL-23in the culture supernatants were measured by ELISA.Results1. The DNA sequences of the cloned malE gene were completely identical with the reported corresponding gene. The constructed prokaryotic expression system could express MBP. Then, MBP was identified by Western Blot.2. MBP promoted the expression of CD40, CD54, CD80, CD86and MHC I on the surface of DCs, while inhibited the expression of MHC II.The mean fluorescence intensity (△MF1) of CD40, CD54, CD80, CD86, MHC-I and MHC-II on the surface of DC2.4was detected by flow cytometry, the△MFI were as follows:23.45±1.68,335.15±6.55,3.54±0.36,60.75±4.18,250.15±5.43and64.35±6.02, respectively; after cocultured with1μg/mL MBP, the△MFI were as follows:35.65±1.30,412.15±10.27,5.45±0.20,145.15±6.55,343.15±13.21and43.05±4.15, respectively; after cocultured with5μg/mL MBP, the△MFI were as follows:31.45±1.95,410.15±4.52,5.51±0.20,150.15±7.01,391.15±6.66and25.05±4.42, respectively. The data showed that MBP promoted the expression of CD40, CD54, CD80, CD86and MHC I on the surface of DCs, while inhibited the expression of MHC-II compared with untreated group.3. MBP stimulated the DCs to secreting IL-23, but did not affect the expression of IL-1β, IL-6and IL-12by DC significantly.The concentrations (pg/mL)of IL-β, IL-6, IL-12and IL-23of untreated DC2.4in culture supernatant were1025.50±35.36,311.9±27.02,18.4±1.15and2.3±2.08, respectively; after treated with1μg/mL MBP, the concentrations (pg/mL) were937.00±53.03,332.8±1.09,16.5±0.86and26.4±2.08, respectively; after cocultured with5μg/mL MBP, the concentrations (pg/mL) were945.50±21.21,311.13±21.58,16.5±2.29and26.4±2.12, respectively. The data indicate that MBP stimulate the secretion of IL-23of DCs compared with untreated group, while IL-1β, IL-6and IL-12have no significant change.ConclusionsMBP might promote endogenous antigen presenting and inhibit exogenous antigen presenting of DC. In addition, DC could be promoted secreting cytokines IL-23which might facilitate the differentiation, amplification and survival of Th17subsets.