Expression Of Human TSHR A Subunit In E.coli And Analysis Of Its Immune Activity

Human thyrotropin receptor A subunit?TSHR A?has important significance in the production of thyrotropin receptor antibody?TRAb?,detection of serum thyrotropin receptor antibody in Graves'patients and immunotherapy for Graves'disease.Given that the natural TSHR A subunit is not available,the yield of the fusion protein obtained by eukaryotic expression is minimal.Obtaining sufficient TSHR protein A subunits has become a hot and difficult point in the diagnosis and treatment of Graves disease.Since In this study,molecular biology techniques were used to establish A prokaryotic expression system of TSHR A subunit,and to obtain TSHR A subunit segment in escherichia coli.Objective The prokaryotic expression vector pET102/D-TOPO/TSHR A of the human thyroid stimulating hormone receptor?TSHR?A subunit was constructed and expressed in Escherichia coli,analyzed for its immunological activity.Method Amplification of TSHR A by RT-PCR and directional ligation into vector pET102/D-TOPO,transformed by Escherichia coli BL21 Star^TM?DE3?after identification by PCR,restriction enzyme digestion and sequencing.Expression of TSHR A fusion protein induced by IPTG,bacteria are pyrolysised by ultrasonic crusher,identification by SDS-PAGE and Western blotting.After denaturing,renaturing,and nickel column purification,the target protein is combined with Graves patient's serum to iden-tify its immune activity.Results Construction of pET102/D-TOPO/TSHR A expression vector,optimize fu-sion protein expression conditions,identification of recombinant plasmids by SDS-PAGE to express specific proteins of 47 kD Size,Western bloting identified it can be recognized by His antibody,murine anti-TSHR polyclonal antibody,repeated experimental renaturation.After renaturation,the fusion protein showed strong positive binding to the mixed serum of Graves patients,whereas normal human serum did not bind in any form.Conclusion pET102/D-TOPO/TSHR A subunit expression vector The TSHR A fusion protein expressed in E.coli can be used as an antigen for detecting thyrotropin receptor antibodies in the serum of Graves disease patients.