Construction of Prokaryotic Plasmid of Mycobacterium Tuberculosis Heat Shock Protein 70 and Characterizing Its Antitumor Role / 实用儿科临床杂志

ABSTRACT

Objective To construct recombinant prokaryotic plasmid DNA encoding mycobacterium tuberculosis (TB) heat shock protein 70 (HSP70) - Dnak gene obtained from pMT70, then purify its characterizes and the role of rHSP70 in tumor therapy. Methods Amplified by polymerase chain reaction (PCR) and with terminal modification, the Dnak gene was cloned into the vector with T7 promoter and was confirmed by restriction endonuclease digestion. So a new plasmid pMT70 - 3 was constructed and transformed in E. Coli. Strain BL21DE3. The engineering bacteria were induced by IPTG. Western-blot,natine-page and scanning were used to analyze the results. The human HSP70 were induced with peptides of lymphocytic leukemia cells of L1210 of mice in binding buffer which contain MgCl2 and ADP at 37 ℃ , then mice were given rHSP70 peptide - complex by SV injection twice at weekly intervals. Results The Dnak gene could be highly expressed in E. Coli as soluble protein. The expressing efficiency was 65% of the total cell protein and the soluble protein was 90% of the expressed protein. The rHSP70 - peptide complex could improve the immune protective function which had significant difference compared with controls (P