Induction Of Antigen-specific Immune Response By Ova-Hsp70L1 Fusion Protein And Its Related Mechanisms

Heat shock proteins (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 trafficking, and degradation. HSP is a kind of potent adjuvant to induce significant immune responses to associated antigens. Some members of HSP family such as Hsp70 participate in the immune responses including antigen presention and T cell activation. It has been shown that immunization with Hsp70/antigen complex could potently induce antigen-specific CD8+ cytotoxic T lymphocytes (CTL). As a new kind of adjuvant, HSP has attracted much attention and is now regarded as an important molecule in mediating tumor or viral specific immunotherapy.Evidences have showed that Hsp70 prepared from tumor cells or virus infected cells are capable of eliciting potent antigen-specific CD8+ CTL response. The mechanism of Hsp70 mediated cancer immunotherapy is based on the adjuvant effects of Hsp70 and its ability to bind antigen-peptide. Several clinical trials of HSP-peptide complexes with cancer patients have been initiated. More recently, immunization of mice with gp96 or Hsp70 fused with proteins including specific CTL epitopes has been shown to elicit protective immunity. These findings demonstrated the adjuvant effects of Hsp70 and encouraged the potential application of Hsp70 fusion proteins in vaccination.We have previously shown that Hsp70Ll, a new member of HSP70 subfamily cloned from human (dendritic celll (DC) cDNA library, named as Hsp70-like protein l(Hsp70Ll), can be used as an adjuvant-free carrier to stimulate the Thl cellular response to a T cell epitope peptide that is noncovalently linked to the Hsp70Ll. The special properties of hsp70Ll prompted us to investigate whether soluble Hsp70Llfusion proteins could be utilized to elicit MHC class I-restricted CD8+ CTL.We show here that Hsp70Ll fused with a large fragment of chicken ovalbumin (ova) could stimulate to produce ovalbumin-specific Thl response in H-2b mice. The experiments will be described in three parts as follows. Part I: Construction, expression, and purification of recombinant proteinsThe DNA fragment encoding amino acids 161-276 of ovalbumin ( ova ) was cloned by PCR from plasmid pcDNA3-OVA containing the full ovalbumin coding sequence as a template and inserted into the pET24a+ expression vector (recombinant vector designed as pET24a- ova) to produce ova protein with a 6xHis tag at the C-terminus, including a translation stop codon immediately after amino acid 276 of ovalbumin.The DNA fragment encoding Hsp70 was cloned by PCR from plasmid pcDNA3-Hsp70 containing the full Hsp70 coding sequence as a template and subcloned into the pET24a+ expression vector(recombinant vector designed as pET24a-Hsp70) to produce ova protein with a 6xHis tag at the C-terminus. Plasmid pET24a- ova -Hsp70 was created by subcloning ova and Hsp70 into pET24a (pET24a- ova -Hsp70) to produce ova-Hsp70 fusion protein with a 6xHis tag at the N-terminus.The full encoding region of Hsp70Ll cDNA without translation stop codon was cloned by PCR from plasmid pQE30-Hsp70Ll containing the full Hsp70Ll coding sequence as a template and subcloned into the pQE30 vector with ova sequence at the 5'-terminus (pQE30- ova -Hsp70Ll) to produce fusion protein with a 6xHis tag at the N-terminus.All plasmids were identified by sequencing in both directions with double-stranded DNA templates and proteins were obtained through prokaryotic expression induced by isopropyl- B -D-thiogalactopyranoside(IPTG). pQE30- ova -Hsp70Ll plasmid was confirmed and then transformed into competent cells of Ml 5 (pREP4). ova -Hsp70Ll expression in Ml 5 cells was induced in 2YT medium by IPTG Recombinant proteins with molecular weight of 68 kD was observed mainly inthe inclusion bodies. pET24a-ova ^ pET24a-Hsp70 and p...