Prokaryotic expression of BTBD10 and preparation of its polyclonal antibody

Objective To clone and express the BTBD10,and prepare the rabbit polyclonal antibody against BTBD10. Methods Full length BTBD10 gene was amplified by PCR and cloned into the expression vector pET32a to construct recombinant expression plasmid pET32a-BTBD10.The recombinant plasmid was transformed into E.coli ROSSET and induced to express recombinant protein with IPTG.The fusion protein was further purified by affinity chromatography.A rabbit was immunized with the purified BTBD10 fusion protein to produce polyclonal antibody,and the production of antibody was identified by Western blot. Results Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid contained the correct BTBD10 code reading frame.SDS-PAGE demonstrated that the induced IPTG expressed the fused protein at 85kU,which was consistent the expected molecular weight.Western blot showed that the antibody showed a good specificity and a high titer at 56kU on the target band. Conclusion The BTBD10 prokaryotic expression plasmid has been successfully constructed,with highly-purified recombinant BTBD10 protein and rabbit polyclonal antibody against BTBD10 obtained.