| Thl cellular immune response is crucial for anti-tumor and anti-microbial immunity. The use of powerful immunomodulatory adjuvants to induce Thl polarization is important in vaccination strategies. However, traditional adjuvants, such as aluminium salts and oil emulsions, mainly evoke Th2 response characterized by antibody production to conformational determinants of antigen. New generation of adjuvants including CpG-rich motifs, monophosphoryl Lipid A (MPA) and Quil A saponin (QS21) exhibit adjuvant effects with Thl polarization. It is assumed that these effects result from the activation of antigen-presenting cells (APC), especially dendritic cells (DC). In recent years, heat shock protein (HSP), a kind of chaperone molecule, being revealed to interact with APC and has potent adjuvant capability in the induction of antigen-specific CD8+ cytotoxic T lymphocyte (CTL) and Thl response, has attracted much attention.HSPs are the most highly conserved proteins in prokaryotes and eukaryotes. Historically, HSPs were regarded as stress-induced proteins that showed higher expression after stimulation with ultraviolet radiation, heat shock, or viral or bacterial infection. In the cell, HSPs are molecular chaperones performing vital functions in protein synthesis, folding, and translocation. However, recent studies have documented that extracellular HSPs can interact with APC, especially DC, and exhibit potent adjuvant functions in stimulating the host immune response. Interaction of HSPs and APC leads a cascade of events including re-presentation of the chaperoned peptides by the major histocompatibility complex (MHC), secretion of proinflammatory cytokines, and maturation of DC. These properties make HSPs a kind of potent adjuvant to induce significant immune responses to associated antigens. The HSP70 subfamily is one of the most important HSP subfamilies, characterized by ATPase activity and with a relative molecular weight of 70 kDa. Evidences haveshowed that HSP70 and gp96 prepared from tumor cells or virus infected cells are capable of eliciting potent antigen specific CD8+ CTL response. It is revealed that HSP70 can bind to CD91, CD 14, TLR2/4 receptor on the surface of APC thus HSP70 can activate APC and facilitate the representation of HSP-associated antigen via a TAP dependent or TAP independent route by APC. These findings demonstrated the adjuvant effect of HSP70 and encouraged the potential application of HSP70 in vaccination.We report here a new member of HSP70 subfamily cloned from human DC cDNA library, named as HSP derived from DC (HSP-DC). As DC are the most important APC which play critical roles in the initiation of immune response, our research focused on the interaction of recombinant HSP-DC and DC. To reveal the functional novelty of HSP-DC, we compared HSP-DC to HSP70 in the experiments. In order to evaluate the adjuvant effect of HSP-DC in vivo, we immunized C57BL/6 mice with HSP-DC-OVA257-264 hybrid, detected the induction of OVA257-264-specific Thl responses, CTL and anti-tumor immunity. The findings will be described in four Parts as follows.Part I: Expression and purification of recombinant HSP-DC protein. Recombinant HSP-DC protein was obtained through prokaryotic expression. The full encoding region of HSP-DC cDNA was inserted into the pQE30 expression vector (recombinant vector designed as pQE30-HSP-DC) to produce recombinant HSP-DC protein with a 6×His tag at the N-terminus. Confirmed pQE30-HSP-DC was then transformed into competent cells of Ml 5 (pREP4). HSP-DC expression in Ml5 cells was induced in 2YT medium by isopropyl- β -D-thiogalactopyranoside(IPTG). A recombinant protein with a molecular weight of 56 kD was observed mainly in the inclusion bodies. To obtain LPS free protein, the inclusion bodies were washed extensively, and then solubilized with urea. His-tagged recombinant HSP-DC was purified by His-Trap metal chelation chromatography and DEAE column. The purity of purified HSP-DC fusion protein was >97% as confirmed by SDS-PAGE analysis. LPS contamination was <... |
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