Preparation Of Hsp70-Peptide Complexes Derived From Human Gallbladder Cancer Cells And Its Effects Of Immunostimulation On Human Peripheral Blood Mononuclear Cells

Objective To construct eukaryotic expression vector pcDNA3.0-hsp70 and transfect SGC996 gallbladder cancer cells. HSP70-SGC996 peptide complexes were purified to study the immunoactivating effects on human peripheral blood mononuclear cells and to explore its anti-tumor immune responses. Methods The eukaryotic expression vector pcDNA3.0-hsp70 was constructed by introducing hsp70 DNA fragment into the sites of EcoR I and BamH I of pcDNA3.0 vector. DNA sequencing and restriction endonucleases were used to test the recombinant vector. The expression vector pcDNA3.0-hsp70 was introduced into SGC996 cells by liposome-medium transfection. The positive clones were selected in the growth medium supplemented with G418. Four representative clones were expanded and screened for HSP70 production by Western blot and flow cytometry using anti-HSP70 antibody. The HSP70-SGC996 peptide complexes were extracted and purified from the cells of Overexpression clone by ADP-affinity chromatography. The immunostimulating effect of the HSP70-SGC996 peptide complexes on human PBMCs were assayed by ELISA and flow cytometry. Results The eukaryotic expression vector pcDNA3.0-hsp70 was constructed and transfected successfully into SGC996 gallbladder cancer cells. Single positive clone was picked out by limited dilution and cultured amplificationally. Expression levels of HSP70 increased significantly in the transfected SGC996 cells. The HSP70-SGC996 peptide complexes purified by ADP-affinity chromatography exhibited a 70KD protein band by SDS-PAGE and Coomassie blue staining. The purity and yield of HSP70 reached 100% and 26.8% respectively. Human PBMCs could be activated with HSP70-SGC996 peptide complexes and secreted high-level IFN-γat 48h after treatment (P<0.05). Significant difference could be seen between modified groups and control group. Concentration of the complexes seems no linear correlation to PBMCs activation. Low concentration complexes (1μg/ml) could produce stronger stimulation to PBMCs than other groups. Immunophenotype analysis of human PBMCs showed that CD25 and HLA-DR positive B lymphocytes, CD25 positive T lymphocytes, CD86 positive PBMCs increased remarkably after treated by HSP70-SGC996 peptide complexes, which meant that T and B lymphocytes were effectively activated. Conclusions The eukaryotic recombinant expression vector pcDNA3.0-hsp70 was successfully constructed and transfected into SGC996 gallbladder cancer cells. The stable transfection clones were acquired and expressed high level HSP70. The HSP70 bound peptide complexes could be isolated and purified by ADP-affinity chromatography. The HSP70-tumor antigen peptide complexes could effectively stimulate the human PBMCs, activate B lymphocytes to enhance the antigen presentation and increase cytokines secretion of T lymphocytes. This study laid a foundation for the further research of the function of HSP70 in immunotherapy of human cancer.