[Preparation and identification of the polyclonal antibody against heat shock protein 70 (DnaK) from Shigella flexneri 5a M90T].

OBJECTIVE

To generate polyclonal antibodies against Shigella flexneri 5a M90T heat shock protein 70 (DnaK) and identify the specificity and titer of antibodies.

METHODS

The sequence of Shigella flexneri 5a M90T dnaK gene was amplified by PCR and inserted into expression vector pET-24a to obtain recombinant plasmid pET24a-dnaK. Then the recombinant plasmid pET24a-dnaK was transformed into E.coli BL21 (DE3) and expressed under IPTG induction. The recombinant DnaK proteins with His-tag (DnaK-His) were then purified by affinity purification. The polyclonal antibodies were prepared by immunizing the mice with the purified recombinant proteins and then harvesting mouse sera. The specificity and titer of the polyclonal antibodies were identified by Western blotting, ELISA and immunofluorescent staining.

RESULTS

The recombinant plasmid pET24a-dnaK was expressed efficiently in E.coli BL21 (DE3). The polyclonal antibodies harvested from the mice immunized with DnaK-His were demonstrated being specific to Shigella flexneri 5a M90T DnaK proteins, and could be efficient in Western blotting, ELISA and immunofluorescent staining.

CONCLUSION

The polyclonal antibodies against Shigella flexneri 5a M90T DnaK protein have been successfully prepared.