Prokaryotic expression of F10 and preparation of its polyclonal antibody

Objective: To induce the expression of F10 in E.coli and prepare the rabbit polyclonal antibody against it.

Methods: The gene of F10 was amplified by PCR, and cloned into expression vector pET-GST to construct recombinant expression plasmid pET-GST/F10. The recombinant plasmid was transformed into E.coli BL21 and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and analyzed by SDS-PAGE and Western blot. A rabbit was immunized with the purified F10 fusion protein to produce polyclonal antibody, and the production of antibody was confirmed by ELISA.

Results: Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid contained correct coding region of F10. SDS-PAGE demonstrated that the recombinant protein was expressed with the expected molecular weight at 61 kD. After purified, the purity of the fusion protein was above 90%. Western blot confirmed the recombinant protein was GST/F10 fusion protein. Rabbit polyclonal antibody was obtained, the titer of which was 1:20 000.

Conclusions: F10 recombinant expression vector has been successfully constructed and F10 protein has been expressed. The obtained rabbit anti F10 antibody has a high titer and will facilitate the study of the biological function of F10.