Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Cloning of Rat Thyroid Hormonr Receptor β1 cDNA and Its Expression in E.coli.

Author MuCheng
Tutor ZhangJingYu;GuoGang;LiangDongChun;WangBaoLi
School Tianjin Medical University
Course Biochemistry and Molecular Biology
Keywords TR gene engineering fusion expression EMSA RLBA
Type Master's thesis
Year 2006
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Thyroid hormone receptor (TR) is member of nuclear receptor superfamily and expressed in almost every tissue in normal body.TR has two subtypes,TR α and TR β .Because of different transcript start position and different splice,each subtype has several isoforms,including TRal,TRa2,TRa3,TRΔal,TRΔa2,TRβl, TRβ2,TRβ3,TRΔ63,etc.In clinic research,TR gene has a close relationship with resistance to thyroid hormone (RTH).And thorough study of TRβstructure is also very important for design of new blood lipid-lowering drugs.In this study,firstly,full-length sequence encoding rat TRβ1(rTRβ1) gene was obtained by RT-PCR using total RNA derived from rat liver as template, and thenit was cloned into pGEM-T Easy vector.Secondly,after corrected the wrong nucleotide,the target sequence was inserted into prokaryotic fusion expression vector pQE-30Xa,tested by DNA sequencing.Finally, recombinant plasmid pQE-30Xa/rTRβ1 was transformed into E.coli M15[pREP4] and expressed.The amount and molecular weight of the recombinant protein were tested by SDS-PAGE.Western blotting revealed that the 56-KD fusion protein was rTRβ1.After the culturing conditions were optimized,the yield of rTRBl was 6mg/L and accounted for 24.32% of total protein in E.coli M15[pREP4].The target proteins containing 6 X His affinity tag facilitates binding to Ni-NTA resin in metal chelating affinity chromatography.After the proteins binding non-specifically to resin were removed by washing solutions,the target proteins were eluted from resin with apparent higher concentration of imidazole, dialyzed and concentrated by freeze-dry.By electrophoretic mobility shift assay (EMSA) and radioligand binding assay(RLBA), recombinant rTRB1 was identified to have biological activity of binding target DNA and T3.

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