Construction Of The Fusion Protein Of HTERT And HSP70 And Its Effect On DC Differentiation And Maturation

Since 1970s, the morbility and mortality of malignant tumors have been showing a tendency to increase. Cancer has become the first cause of death in urban and rural areas in our country. Presently, surgery, chemotherapy and radiotherapy are the main therapeutic methods. With the increasing development of biotechnology, immunotherapy has been the main topic of research. This study is to construct a fusion protein of human telomerase reverse transcriptase (hTERT) and heat shock protein 70(HSP70), and to investigate the effect of the fusion protein on DC differentiation and maturation.Objective To construct the prokaryotic expression plasmid of hTERT and HSP70 fusion gene, to detect the expression of the fusion gene in E. coli BL21, to purify the fusion protein, and to observe the effect of the fusion protein on DC differentiation and maturation. Methods The gene fragment of hTERT(532aa～637aa) and the whole cDNA of HSP70 were amplified by PCR, then they were digested by endonucleases and cloned into the prokaryotic expression plasmid pET-32a to construct the recombinant vector pET32a-hTERT and pET32a-HSP70. Then the pET32a-HSP70 was digested and the HSP70 gene segment was cloned into the 3'-terminal of the pET32a-hTERT to construct the prokaryotic expression plasmid pET32a-hTERT-HSP70. The E. coli BL21 containing the vector pET32a-HSP70 or pET32a-hTERT-HSP70 was induced by IPTG and the fusion proteins were purified. The DCs loaded with the fusion proteins were harvested on the 10th day and the surface molecules were detected by flow cytometry.Results The DNA sequencing showed that the prokaryotic expression vectors pET32a-HSP70 and pET32a-hTERT-HSP70 were successfully constructed. After being induced by IPTG; the E. coli BL21 containing the plasmid pET32a-HSP70 or pET32a-hTERT-HSP70 could express 90kD or 110kD protein respectively. Purified soluble fusion proteins with His-tag were obtained. After being loaded with the fusion proteins HSP70 and hTERT-HSP70, CD80, CD86 and HLA-DR of DCs were up-regulated, and CD14 was down-regulated. Conclusion The expression vectors of hTERT and HSP70 fusion gene were successfully constructed, and the purified fusion proteins were obtained, and they could enhance the differentiation and maturation of DCs. This will provide the foundation for the further research associated with the fusion proteins of HSP70 and hTERT-HSP70.