[Prokaryotic expression of mouse Stra8 and preparation and identification of rabbit anti-mouse Stra8 polyclonal...

Objective To prokaryotically express mouse stimulated by retinoic acid gene 8 (Stra8) and prepare rabbit anti-mouse Stra8 polyclonal antibody. Methods The recombinant plasmid pET28a-Stra8 was constructed by cloning technology and identified by double enzyme digestion and sequencing, and then transformed into E. coli BL21 (DE3). The expression of Stra8 recombinant protein was induced by IPTG. The prokaryotic protein was purified by His-TAG affinity chromatograph and then used to immune New Zealand white rabbits to obtain Stra8 polyclonal antibody. ELISA, Western blot and immunofluorescence assays were used to determine the antibody titer, validity and specificity, respectively. Results The recombinant plasmid pET28a-Stra8 was constructed successfully as double enzyme digestion and sequencing showed, and the prokaryotic protein was expressed and purified effectively. The titer of the polyclonal antibody reached 1:10⁶. Western blotting showed that the polyclonal antibody could specifically recognize native Stra8 protein in the testis. Immunofluorescence assay revealed that the polyclonal antibody had good reactivity, and could recognize Stra8 protein in mouse testis. Conclusion Stra8 prokaryotic protein can be effectively induced in E. coli and specific rabbit anti-Stra8 polyclonal antibody has been prepared.