Generation of the Antibody Selectively Against a Novel Thyroid Hormone Receptor Beta Isoform

A novel thyroid hormone receptor isoform TR beta Delta was newly reported and deposited in Genbank(DQ191165.1) by our lab. To gain more understanding of this new variant. We raised rabbit polyclonal antibodies specifically against the characteristic epitope, amino acids 129-164 named as betaDelta36, of TRbetaDelta. Coding sequence for this betaDelta36 peptide was obtained by PCR amplification and inserted into a GST fusion expression vector pGEX-3X. Recombinant plasmid was transformed into E. coli DH 5alpha and induced expression by 1 mmol/L IPTG at 25 degrees. Glutathione Sephorase 4B affinity chromatography purified fusion protein GST-betaDelta 36 was applied as immunogen to immune rabbits. An ELISA was developed to determine the titer of the antiserum. Rabbit anti GST-betaDelta36 antiserum was collected when the titer reached 1:12000, and serum IgG was further purified by protein A affinity chromatography. Western blot was applied to testify the specificity of the antibody. The results showed that this rabbit anti-GST-betaDelta 36 polyclonal antibody could selectively recognize recombinant TRbetaDelta protein, but not cross react with recombinant TRbeta1.