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[Prokaryotic expression of Drosophila selenoprotein D-SelK and preparation of polyclonal antibody to D-SelK].

AIM
To clone and encode Drosophila selenoprotein D-SelK structure gene, express it in E.coli efficiently, and after purification, to generate its antibody in rabbits.


METHODS
D-SelK gene segment amplified from pGM-T-D-SelK plasmid by PCR was inserted into pGEX-6p-1 to construct recombinant plasmid pGEX-6p-1-D-SelK. The recombinant plasmid was transfected into E.coli BL21(DE3) to express the recombinant protein D-SelK in E.coli under IPTG induction. The protein was purified by denaturation and electrophoresis, and then identified by SDS-PAGE and Western blotting. Polyclonal antibody to D-SelK was obtained by immunizing rabbits with the protein. Quality and quantity of the antibody was examined.


RESULTS
D-SelK gene segment was successfully inserted into pGEX-6p-1 and the positive clones of the recombinant plasmid was identified by PCR screening and restriction endonuclease analysis. The target protein was effectively expressed in E.coli by the IPTG induction. Through immunizing rabbits with the purified target protein, we obtained the specific antibodies to D-SelK, the titer of which was more than 1:51 200. The polyclonal antibody had a good specificity to D-SelK.


CONCLUSION
D-SelK recombinant protein and rabbit anti-D-SelK polyclonal antibody with high specificity were obtained, which provides good tools for further research on the functional characterization of D-SelK.