Abstract:

Objective     To clone the outer membrane protein OprF of Pseudomonas aeruginosa(PA), and to construct and identify a prokaryotic expression plasmid. Methods    The DNA genome was extracted from PA, and the carboxyl terminus of the OprF gene was amplified by primers of PCR. The recombinant plasmid pET28b-F was constructed by cloning the OprF cDNA into the prokaryotic expression vector pET28b. After being identified by restriction endonuclease digestion analysis and DNA sequencing, the pET28b-F was transformed into E. coli BL21 and induced by IPTG. The expressed protein was analyzed by SDS-PAGE and Western blot. Monoclonal antibodies(McAbs) against OprF were prepared by the hybidoma technique and screened by ELISA. Results    The construction of the recombinant expression plasmid pET28b-F was correct by restriction enzyme, PCR and DNA sequencing. Then the expression plasmid expressed a corresponding protein OprF after induction of IPTG. Four McAbs could specifically combine with the OprF protein of PA. Conclusion    Successful construction of OprF in the prokaryotic expression vector and specific McAbs established with this method can provide the basis for further research into preparing the specific antibody to OprF and into conveniently detecting PA in wound infection.

Key words:  Pseudomonas aeruginosa;  Prokaryotic cells; Antibodies, monoclonal; Membrane Proteins