The preparation of human HSP73 antibody and the studies on expression of HSP70 in tumor cell lines after hyperthermia treatment

Objective To prepare the special antibody of human heat shock cognate protein 73 (HSP73) and to study the effects of Tween 80 combined with 41℃ heating on HSP70 expressions in BGC 823 gastric cell line and B16 melanoma cell line.Methods Human HSP73 gene was inserted into plasmid vector pET3c to construct a recombinant plasmid,pETHSP73,which transformed E.Coli strain BL21.BL21 expressed human HSP73 under induction of IPTG.The protein was extracted and purified by Electrophoresis and Affinity Chromatography.The polyclone antibody of HSP73 was obtained from immunizing New Zealand rabbits with HSP73.The antibody was used to assay the expressions of HSP70 in BGC 823 cells and B16 cells after treatment with Tween 80 and 41℃ hyperthermia.Results The pETHSP73 could express high level HSP73,a molecular weight 73kD protein,in Ecoli BL21.Western blotting showed the protein took on the antigen speciality of human HSP70.The analyses of amino acid ratio and N terminal sequence of the protein coincided with those of the related reports.The dilution of prepared HSP73 antibody was over 1∶8000.The study of immunocytochemistry showed that the expression of HSP70 in both tumor cells was significantly inhibited by Tween 80 in combination with 41℃ hyperthermia.Conclusion pETHSP73 is a high expression vector of HSP73 protein,which keeps biologic activity.The prepared antibody from HSP73 shows a high activity of anti HSP70 in human and mouse.The results suggest that Tween 80 combined with 41℃ heating can significantly inhibit the expression of HSP70 in tumor cells,thus decrease thermotolerance of tumor cells.