[Preparation and application of rabbit anti-mouse Setd8 polyclonal antibody].

Objective To purify the recombinant Setd8 protein and prepare rabbit anti-mouse Setd8 polyclonal antibody. Methods The recombinant plasmid pET-30a-Setd8 was constructed by double enzyme digestion and linkage, and then transformed into E.coli BL21. The expression of the target protein was induced by IPTG and the expression product was purified by Ni-NTA affinity chromatograph. The purified protein was used to immunize New Zealand white rabbits to produce polyclonal antibody. The titer and specificity of the antibody were identified by ELISA, Western blotting and immunohistochemistry. Results The prokaryotic expression vector pET-30a-Setd8 was constructed successfully. After induced by IPTG, the recombinant Setd8 protein was expressed effectively in E.coli BL21. Polyclonal antibody against Setd8 was generated by immunizing rabbits with the routine method. ELISA showed that the titer of rabbit anti-Setd8 antiserum was 1:1 000 000. Western blotting demonstrated that the polyclonal antibody could recognize the native mouse Setd8 protein. Immunohistochemistry revealed that Setd8 protein recognized by the polyclonal antibody was mainly distributed in the nucleus of spermatogonia in adult mouse testis. Conclusion Using the prokaryotic expression vector pET-30a-Setd8, we have prepared successfully the polyclonal antibody with high affinity and specificity.