[The expression of humam sperm actin-like protein 7a and preparation of its polyclonal antibody].

OBJECTIVE

To express and purify the recombinant human sperm actin-like protein 7a (ACTL7a) N terminal (1-70) in E.coli, and prepare the corresponding rabbit anti-ACTL7a polyclonal antibodies.

METHODS

We constructed the recombinant expression plasmid pGEX-6p-1-ACTL7a (1-70), transformed the plasmid into E.coli BL21 (DE3), and selected positive recombinant strain. The target protein was induced to express by IPTG and purified by nickel ions-chelating resin and glutathione sepharose 4B resin, and finally obtained by molecular sieve chromatography. The purified ACTL7a (1-70) protein were used to immunize New Zealand rabbits to prepare anti-ACTL7a polyclonal antibodies. The antibody titers were detected by ELISA, the antibody specificity by Western blotting, and the localization of ACTL7a in the spermatogenic cells of seminiferous tubule epithelium by immunofluorescence histochemistry.

RESULTS

The target protein ACTL7a (1-70) was successfully expressed by IPTG in E.coli, and the corresponding antiserum was obtained in the rabbits immunized with it. The titer of the antiserum reached 1:160 000 and it was proved with a good specificity.

CONCLUSION

ACTL7a was expressed in the constructed prokaryotic expression vector. After purification and immunization, high titer and specific anti-ACTL7a antibody was prepared in rabbits.