Chaperokine function of recombinant Hsp70-Her2 fusion protein produced in insect cells using a baculovirus expression system Free

A102

Immunotherapies and tumor vaccines are the promising approaches for effective treatment because they can augment antigen-specific immune responses and with fewer side effects than some chemotherapeutic compounds. Extracellular Hsp70 plays a critical role in innate and adaptive immune responses, and it is an ideal adjuvant for development of an effective tumor vaccine. We develop Hsp70-HER2 fusion protein vaccines for their vaccine potential in vivo. Here, we demonstrated its chaperokine function in vitro. In order to control endotoxin contamination, we have taken advantage of baculovirus expression system. The hsp70 gene and a fragment of her2 gene were amplified by PCR and cloned into pBACgus2cp to construct the recombinant bacmids. The recombinant bacmids were constructed by inserting hsp70 gene into HindIII and XhoI sites, and her2 gene fragments into EcoRI and HindIII sites. The recombinant bacmid DNA was co-transfected with linear viral DNA into the Sf9 cells to produce the recombinant virus containing hsp70 and her2 fusion gene to express the target proteins. PCR results showed the recombinant bacmids were constructed successfully. Hsp70 and Hsp70-HER2 fusion proteins expressed in insect cells were assessed by SDS-PAGE and Mass spectrometry. Hsp70 and Hsp70-HER2 fusion proteins stimulated potent calcium flux in human monocytes, a known function of Hsp70 but not endotoxin. These proteins exhibit classical chaperokine functions including stimulation of pro-inflammatory cytokine and chemokines production. The recombinant HSP70-HER2 fusion proteins allow the investigation for new feasible drug targeting HER2 positive tumor cells.