ABSTRACT
Objective: To study the cloning, recombinant expression and purification of human HSP70D. Methods: The full length code region of HSP70D was amplified by RT PCR from HeLa cells. Then the PCR product was inserted into pET 24a(+), by NdeⅠ/XhoⅠ double digestion. The recombinant expression vector pET24a HSP70D was transformed into E. coli strain BL21. Positive clones were determined by NdeⅠ/XhoⅠ digestion and sequence analysis, which was used in the expression and purification of HSP70D protein. The engineered bacteria were collected after fermentation. The recombinant HSP70D was purified by using ethanol precipitation, anion exchanger, hydrophobicity interaction chromatography, and finally high resolution gel filtration chromatography. Results: The expression level of HSP70D was over 20% of total protein in the engineered bacteria. After purification, electrophoretically pure (more than 95%) rhHSP70D was obtained. Conclusion: This study setup a satisfied down stream process which can be used for production of large quantity of recombinant HSP70D.
ABSTRACT
Objective: To determine the adjuvant activity of recombinant human HSP70D protein. Methods: Dendritic cells were cultured in vitro , rhHSP70D was added into the culture medium, the cytokine secretion and maturation of dendritic cells(DC) stimulated by rhHSP70D were measured; C57BL/6 mice were immunized with rhHSP70D OVA 257 264 complexs reconstituted in vitro , and the protective antigen specific immune response against tumor challenge were evaluated in the immunized mice. Results: rhHSP70D stimulated human DC to secrete IL 1?, IL 12 p70 and TNF ?, up regulated the HLA DR,CD86 and CD40 expression on DC and improved the antigen presentation function of DC; After immunization with the hybrid of rhHSP70D and OVA 257 264 , the mice were able to resist the challenge of E. G7 OVA, which was a tumor cell line expressing model antigen OVA. Conclusion: rhHSP70D can induce maturation of DC, immunization of rhHSP70D peptide complexs can induce antigen specific immune response, suggesting that rhHSP70D may be a new adjuvant in immunotherapy of the cancer and infection diseases.