TRBP分子克隆及其功能初步研究

Molecular cloning and preliminary functional study of TRBP

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。

AIM： Construct prokaryotic expression vector carrying mouse TRBP（TAR RNA-binding protein） gene and test the double-stranded RNA binding ability of TRBP.METHODS： RT-PCR was used to obtain TRBP cDNA from mouse genomic DNA.Then,we built the His-tag fusion expression vector of TRBP and transformed it into E.coli BL21（DE3）.Ni-NTA beads were used to isolate and purify the recombinant protein and vitro transcription was used to get Pre-miR-122.Finally,SDS-PAGE and ITC（isothermal titration calorimetry） assay were both used to validate TRBP＇ s binding ability with Pre-miR-122.RESULTS： We purified the recombinant protein TRBP whose molecular weight is 32.4 kDa.The purified bioactive TRBP protein binding on NI-NTA beads showed that it had a strong binding capacity on Pre-miR-122.CONCLUSION： We constructed TRBP prokaryotic expression system successfully and studied the double-stranded RNA binding ability of TRBP preliminarily.