[Molecular cloning and preliminary functional study of TRBP].
OBJECTIVE
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.


CONCLUSIONS
We constructed TRBP prokaryotic expression system successfully and studied the double-stranded RNA binding ability of TRBP preliminarily.
School of Medicine and Pharmacy, Ocean University of China, Qingdao, China. lzj2385759@126.com
Zhan-jun
ZJ
Liu
Ying
Y
Zhu
Wen-gong
WG
Yu
Xin-zhi
XZ
Lu
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000099253:Trans-Activation Responsive RNA-Binding Protein
D035683:MicroRNAs; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000099253:Trans-Activation Responsive RNA-Binding Protein; C544354:Mirn122 microRNA, mouse