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目的:构建TRBP原核表达载体,评价表达的重组TRBP蛋白对双链RNA的结合能力。方法:利用RT-PCR扩增小鼠TRBP基因双链RNA结合区,构建其与His标签融合的表达载体,转化E.coli BL21(DE3)菌株,利用Ni-NTA磁珠对重组蛋白进行分离纯化;体外转录合成小鼠肝脏miR-122前体RNA Pre-miR-122,分别利用PAGE电泳和等温量热滴定仪检测TRBP与Pre-miR-122的结合能力。结果:分离纯化得到的重组TRBP蛋白为可溶蛋白,Mr为32 400,结合在NI-NTA磁珠上的TRBP能有效的结合Pre-miR-122。结论:成功建立了TRBP原核表达系统,初步研究了TRBP重组蛋白与双链RNA的结合能力。
OBJECTIVE: To construct a prokaryotic expression vector for TRBP and evaluate the binding ability of the expressed recombinant TRBP protein to double-stranded RNA. Methods: The double stranded RNA binding region of mouse TRBP gene was amplified by RT-PCR. The recombinant plasmid was fused with His tag and transformed into E.coli BL21 (DE3) strain. The recombinant protein was purified by Ni-NTA magnetic beads The pre-miR-122 of mouse liver miR-122 precursor was synthesized by in vitro transcription. The binding ability of TRBP to Pre-miR-122 was detected by PAGE electrophoresis and isothermal calorimetry respectively. Results: The recombinant TRBP protein isolated and purified was soluble protein with Mr 32 400. TRBP binding to NI-NTA magnetic beads could effectively bind to Pre-miR-122. Conclusion: The prokaryotic expression system of TRBP was successfully established and the binding ability of TRBP recombinant protein to double-stranded RNA was studied preliminarily.