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目的研究B细胞末端分化过程中miR30b新的作用靶点。方法经生物信息学分析,利用特异性引物以及定点突变引物从小鼠c DNA中分别扩增大小为530、361和189bp三个目的片段,胶回收并对361和189bp进行拼接,获得野生型、突变型小鼠Bach2 mRNA特异性片段,分别与pmir GLO载体连接,构建野生型、突变型重组荧光素酶报告基因质粒pmir GLO-m Bach2、pmir GLO-m Bach2 mt;与miR30b共转染至HEK293 T细胞,分析其荧光素酶活性。结果重组荧光素酶报告基因质粒经双酶切及测序证实构建正确;共转染后,与pmir GLO+miR30b组相比,pmir GLO-m Bach2+miR30b组的荧光素酶活性明显降低(P<0.05),而pmir GLO-m Bach2 mt+miR30b组的荧光素酶活性则无明显改变(P>0.05)。结论小鼠Bach2 mRNA是miR30b的靶点。
Objective To study the new target of miR30b in the process of B cell terminal differentiation. Methods After bioinformatics analysis, three target fragments of 530, 361 and 189 bp in size were amplified from mouse c DNA by specific primer and site-directed mutagenesis. The fragments were recovered by gel collection and spliced at 361 bp and 189 bp. The wild-type and mutant Type mouse Bach2 mRNA specific fragment, respectively, and pmir GLO vector was ligated to construct a wild type, mutant recombinant luciferase reporter gene pmir GLO-m Bach2, pmir GLO-m Bach2 mt; co-transfected with miR30b HEK293 T Cells were analyzed for luciferase activity. Results The recombinant luciferase reporter plasmid was confirmed by double enzyme digestion and sequencing. After co-transfection, the luciferase activity of pmir GLO-m Bach2 + miR30b group was significantly lower than that of pmir GLO + miR30b group (P < 0.05), while the luciferase activity in pmir GLO-m Bach2 mt + miR30b group had no significant change (P> 0.05). Conclusion Mouse Bach2 mRNA is the target of miR30b.