Plasmid Construction And Protein Expression Of HTSHR In Prokaryotic System

Objective: To construct the hTSHR prokaryotic expression plasmid,express in the E.coli system to obtain a hTSHR protein with intact structural and immunological activity.Methods: To design primers,and catch the target gene using PCR method.The pET-28a(+)plasmid vector was linearized and the target gene and the linearized vector were ligated by homologous recombination,and the recombinant DNA successfully ligated was transformed into competent cells of the bacteria.The positive clones were identified by colony PCR,and the PCR-positive broth was sequenced and blasted,which is completely coincident with the target gene sequence is a successfully constructed hTSHR prokaryotic expression plasmid.The recombined plasmid pET-28a(+)-hTSHR was transformed into E.coli BL21(DE3)and the recombinant protein was induced by IPTG.The fusion protein was detected by Western blotting using TSHR antibody,His antibody and TRAb positive serum from Graves patients as probes.10% SDS-PAGE electrophoresis and Coomassie brilliant blue staining were used to investigate expression conditions and expression patterns.Results: PCR amplification of the 2366 bp hTSHR cDNA fragment was consistent with the expected length.After the recombinant plasmid was induced to express in E.coli BL21(DE3),TSHR and His specific antibodies were used as probes.Western blotting confirmed that the fusion protein had a significantly enhanced specific protein band at 100 KD.The expressed fusion protein can specifically bind to TRAb-positive patient serum,confirming that it has a certain degree of immune activity.The optimal induction concentration of IPTG was 0.5 mmol/L and induction time was 3 h.The hTSHR protein expressed in E.coli exists in a soluble state.Conclusion: In this study,a pET-28a(+)-hTSHR prokaryotic expression plasmid was constructed,and hTSHR protein was expressed in E.coli,which can specifically bind to TRAb.