Cloning, expression of a truncated HSP110 protein that augments the activities of tumor antigen-specific cytotoxic and apoptosis via tHSP110-peptide complex vaccines

The present study used a genetic engineering method to express a truncated heat shock protein 110 (tHSP110) isoform in Escherichia coli and verified its ability to bind to and present macromolecular antigens. Polymerase chain reaction (PCR) was used to obtain the truncated HSP110 gene, which was expressed in E. coli. The tHSP110 protein was non-covalently coupled to the intracellular domain (ICD) of human epidermal growth factor receptor 2 (HER2/Neu) in vitro to construct the antigen peptide complex tHSP110-ICD, which was identified by a co-immunoprecipitation assay. BALB/c mice were immunized 14-day interval for three times with the HSP110, tHSP110, HSP110-ICD, tHSP110-ICD, HSP110-P_851-859 (a complex formed by full-length HSP110 with a cytotoxic T lymphocyte (CTL) epitope peptide of the Her2/neu ICD) and tHSP110-P_851-859 complexes. Fourteen days after the last immunization, D₂F₂ cells were inoculated into BALB/c mice. The in vivo tumor volume of each group was measured every three days after cell inoculation to evaluate the immunization efficacy of the vaccine in each group. The level of the IFN-γ secreted by activated lymphocytes, the specific CTLs activity was detected. Immunohistochemical staining of bcl-2 and bax were measured on the tumor tissues of each group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the expressed tHSP110 protein was 66 kDa in size. The non-covalent coupling of tHSP110 with ICD and peptide were confirmed by a co-immunoprecipitation assay. The in vivo tumor experiment results indicated no differences in the tumor volumes of the tHSP110-ICD and HSP110-ICD groups. In contrast, the tumor volume of the tHSP110-ICD group was significantly different compared with the tumor volume of the tHSP110-P_851-859 group. After the mice immunized with tHSP110-ICD, tHSP110-P_851-859 complexes, the complexes have potential immunogenicity, and can induce specific CTLs activity and apoptosis in BALB/c mice. As a tumor vaccine to inhibit in vivo tumor growth, the tHSP110 has the same ability to bind macromolecular antigens and activate tumor immune responses as full-length HSP110.