Cloning Of A Novel Exon Of Thyroid Hormone Receptor β Gene And Its Expression In E.coli

The thyroid hormone receptors (TRs) are members of the nuclear receptor super family that includes glucocorticoid receptor, estrogen receptor, pregnancy hormone receptor, and so on. In 1972, Oppenheimer confirmed TR in cellular nucleus of hepatic and nephridial tissue by labeled T₃. The results from the present investigation suggests there are two genes to encode TRs: Trαand TRβ. According to different position of transcription beginning and variable splicing, Trαforms TRα₁, TRα₂, YRα₃, TRΔα₁, TRΔα₂ and TRβforms TRβ₁, TRβ₂, TRβ₃, TRΔβ₃; TRΔα₁,Δα₂,Δβ₃ are brachytmema-pattern of TRα₁,α₂,β₃. We have found a novel isoforms of TR in the Rat liver. The sequence which has been registered in Gene bank and the register number is DQ191165 is named TRβΔ. The cDNA of TRβΔhas additional 108 bp sequence than TRβ1 between exon 3 and exon 4. It is the unique extended- pattern TR isoforms, and extension of its sequence is between exon 3 and 4 of TRβ. It is possible that there is a new exon between intron 3-4.The purpose of this article is to clone and express the 108bp sequence. The expressed 36 amino acids become immunogen to prepare antibody which is special to TRβΔand can detect the disposition and allocation of TRβΔ. We clone and amplify the same novel exon from the genome library and total RNA of the special rat liver which has TRβΔand the genome DNA and total RNA of common Rat liver by polymerase chain reaction and reverse transcriptase-PCR. Subsequently we make the target sequence insert pGEM-T easy vector. After the sequence is confirmed correctly, we cut the pGEM-T easy vector by the restriction enzyme, insert the target gene into the prokaryotic fusion expression vector and construct pGEX-3X/exon recombinant plasmid. After the sequence is confirmed correctly, it is transformed into the E. coli DH5αand expressed, The size and quantity of expression product is tested by SDS-PAGE. The fusion protein could be recognized by anti-GST monoclonal antibody by Western-blotting. We purify the fusion protein from inclusion body to avoid its degradation. After condition of expression is optimized, the yield of recombination protein is 10mg/L medium and attains 21.78% of total protein. After the denaturation and renaturation of inclusion body, it is purified by Glutathione Sepharose 4B affinity chromatograph. Gel analytical system scanning show the purity of the fusion protein is 91.97%. At last, dialyze and conserve the product in -70℃. The GST-fusion has been prepared as immunogen.