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目的在大肠埃希菌中表达并纯化多聚嘧啶序列结合蛋白(polypyrimidine tract binding protein,PTB)-His融合蛋白,并鉴定PTB与HBV转录后调节元件(HBV posttranscriptional regulatory element,HPRE)RNA的体外结合。方法构建PTB-His融合蛋白的原核表达载体,经IPTG诱导表达后,用亲和层析的方法进行纯化。用磁珠结合与Western印迹的方法,验证PTB与HPRE RNA在体外的特异性结合。结果成功构建了PTB-His融合蛋白的原核表达载体,并在体外大量表达后用亲和层析的方法得到了高纯度的PTB-His融合蛋白。磁珠结合沉淀与Western印迹实验证明PTB与HPRE RNA在体外能特异性的结合。结论体外表达的PTB融合蛋白能与HPRE RNA特异性的结合。
Objective To express and purify the polypyrimidine tract binding protein (PTB) -His fusion protein in Escherichia coli and to identify the in vitro binding of PTB to HBV posttranscriptional regulatory element (HPRE) RNA . Methods The prokaryotic expression vector of PTB-His fusion protein was constructed. After induced by IPTG, it was purified by affinity chromatography. The specific binding of PTB to HPRE RNA in vitro was verified by magnetic bead binding and Western blotting. Results The prokaryotic expression vector of PTB-His fusion protein was successfully constructed and the high purity PTB-His fusion protein was obtained by affinity chromatography after it was expressed in vitro. Magnetic beads binding and Western blotting experiments showed that PTB and HPRE RNA can bind specifically in vitro. Conclusion The PTB fusion protein expressed in vitro can specifically bind to HPRE RNA.