TSHR蛋白表达质粒的构建和TSHR蛋白的表达

Plasmid construction and protein expression of TSHR protein

目的 :为获得足量的TSHR蛋白及建立Graves’病的动物模型。方法 :将构建于pCRTM3中的TSHRcDNA经限制性内切酶处理后 ,重新构建于表达型载体PinPointX质粒中 ,经酶切分析和PCR检测 ,并在E coliJM10 9中表达 ,Westernblotting免疫印迹法检测其免疫活性。结果 :证明TSHRcDNA基因正确地重组于PinPointX中 ,并在E coliJM10 9中表达 ,12 %SDS聚丙烯酰胺凝胶电泳测定表达产物TSHR蛋白分子量为 10 7kD ,Westernblotting免疫印迹法证实其具有免疫活性。结论 :已成功地重组了TSHR的表达质粒 ,并得到了有相应免疫活性的TSHR蛋白。

Objective:To acquire sufficient TSHR protein,and establish animal models of Graves'disease Methods:TSHR cDNA which had been constructed in plasmid pCR TM 3 was digested with the restriction enzymes , TSHR cDNA then reconstructed in expression vector PinPoint X The recombinant plasmid DNA was amplified with PCR or digested with BamHⅠ、BglⅡ,then TSHR protein immunoreactivity was detected with Western blotting Results:Only the recombinant plasmid TSHR/PinPoint X 3 that contained TSHR cDNA fragment was discovered,and it was expressed in the cells of E coli JM109 The molecular weight of the recombinant fusion protein was about 107 kD in 12% SDS-PAGE By using Western blotting, it was confirmed that TSHR protein has immunoreactivity Conclusion:The recombinant expression plasmid of TSHR has been successfully constructed, the corresponding immunoreactivity TSHR protein has been acquired