Preparation Of Heat Shock Protein 70 And NY-ESO-1 Peptide Complexes And Its Antitumor Immunological Effect In Vitro

【Objective】To acquire human heat shock protein 70 using prokaryotic expression technology and prepare HSP70-NY-ESO-1 peptide complexes by HSP70 ligate with NY-ESO-1 peptide in vitro. then detect the efficacy in vitro of DC loaded with HSP70-NY-ESO-1 peptide complexeso to kill NY-ESO-1 positive tumor cells, to explore the effective strategies for enhancing antitumor immunity.【Methods】1. The prokaryotic expression, identification and purification of human HSP70: The cDNA fragment encoding HSP70 was amplified by RT-PCR and cloned into PET-30a vector, The recombinant plasmid PET-30a-HSP70 was transformed to E.coli Rosetta(DE3) and expressed protein by IPTG, The expressed protein was confirmed by SDS-PAGE and western blot, then the product of HSP70 was purified by his-tag column.2. Proliferation and identification of dendritic cells from human cord blood: Combined with rhGM-CSF and rhIL-4 in cultured human umbilical cord blood DC, and then induce DC to be mature with TNF-α. The morphology of DC was observed by inverted microscopy, optical microscopy and transmission electron. The phenotype of DC was analyzed by flow cytometry. The function of DC was identified by mixed lymphocyte reaction.3. The killing efficacy of DC loaded with HSP70-NY-ESO-1 peptide complexes in vitro: acquirement of HSP70-NY-ESO-1 peptide complexes by HSP70 ligate with NY-ESO-1 peptide in vitro. Lymphocytes were stimulated by DC loaded with HSP70-NY-ESO-1 peptide complexes, DC loaded with NY-ESO-1 peptide, DC loaded with HSP70 and DC were not loaded respectively. and the lymphocytes were not stimulated by DC As the control group, In total, there are five groups. lactic dehydrogenase(LDH) release method to detect the capacity of the five groups of lymphocytes to kill NY-ESO-1 positive glioma cells in vitro.【Reasults】1. The prokaryotic expression, identification and purification of human HSP70: Result showed that a fragment about 2000bp was amplified by RT-PCR. The HSP70 gene was cloned into PET-30a identified by enzyme digestion and DNA sequencing; Under the inducement of IPTG, a protein with Mr 72KD identified by SDS-PAGE was expressed by the E coli that contained the recombinant plamid, and could be recognized by anti-HSP70 antibody. the purity of HSP70 protein after purification was more than 95% .2. Proliferation and identification of dendritic cells from human cord blood: The eighth day of mDC possessed morphological characteristic of typical DC. The appearance was irregular, and presented various length. The reasults of flow cytometry about cell surface molecules including HLA-DR,CD86 and CD83 of immature DC and mature DC were respectively 25.20%,17.58%,1.50% and 80.65%,76.59%,74.81%, Mixed lymphocyte reaction confirmed mDC can stimulate a strong mixed lymphocyte reaction in vitro, and imDC were not.3. The killing efficacy of DC loaded with HSP70-NY-ESO-1 peptide complexes in vitro: The lymphocytes immunized with DC loaded with HSP70-NY-ESO-1 peptide complexes were remarkably higher than the other four groups to kill NY-ESO-1 positive glioma cells (p<0.001), and The group of DC loaded with NY-ESO-1 peptide were remarkably higher than The group of DC loaded with HSP70, the group of DC were not loaded and the control group (p<0.001), while the rest of three groups have no significant difference(p>0.05).【Conclusion】1. Successfully constructed a prokaryotic expression vector of HSP70, and highly expressed of HSP70 protein, after purification we can get a lot of high purity HSP70 protein.2. Successfully cultivated a lot of mDC from human cord blood mononuclear cytes with the united utilization of the cell factors of rhGM-CSF, rhIL-4 and TNF-α.3. HSP70-NY-ESO-1 peptide complexes Can active and amplify tumor specific cytotoxic T lymphocyte effectively, and HSP70 can assist NY-ESO-1 peptide in killing NY-ESO-1 positive glioma cells, then enhanced the anti-tumor immunity of NY-ESO-1 peptide.