Revista Facultad de Medicina de la Universidad Nacional de Colombia
Print version ISSN 0120-0011
CELY CASTRO, Rocio et al. PRODUCTION OF RECOMBINANT HEAT SHOCK PROTEIN HSC70 IN Escherinchia coli BL21(DE3) TO OBTAIN POLYCLONAL ANTIBODIES. Rev.fac.med.unal [online]. 2006, vol.54, n.3, pp. 156-168. ISSN 0120-0011.
Background. Live organisms respond against stressing conditions raising the production of stress proteins. The heat shock stress is a very well kwon condition that produces heat shock proteins. Objective. In this work we determined the optimum conditions to produce HSC70 protein in E. coli BL12 (DE3) under stress conditions, and the best way to obtain its polyclonal antibodies. Materials and methods. The best concentrations for isopropyl- ß -D-thiogalactopyranoside (IPTG), bacterial inoculum, type of vector plasmid used pET-3a and pET28a (+) and recovery method of soluble and insoluble forms of HSC70 protein and its polyclonal antibodies were established. Results .Five-colony bacteria inoculum, 2 mM IPTG and sonication treatment were found as best conditions with either expression plasmid. We also produced polyclonal antibodies against the HSC70 protein by inoculating rabbits with this purified protein. Conclusions.Specificity and titer of antiserum was determined by ELISA, Western blotting and immunofluorescence assays. Antiserum was also tested by its inhibition ability of rotavirus infection.
Keywords : antibody formation; HSC70 heat-shock proteins; Escherichia coli.