小鼠肌肉组织特异性微小RNA miR-133a腺病毒表达载体的构建及表达

Construction and identification of recombinant adenovirus expressing murine muscle-specific microRNA miR-133a

目的制备小鼠肌肉组织特异性微小RNA miR-133a的腺病毒表达载体并观察其在HeLa细胞的表达。方法利用PCR技术从小鼠基因组DNA扩增miR-133a前体对应的基因组片段,克隆到质粒pAdTrack-CMV,而后以同源重组的形式插入到腺病毒表达载体,由人胚肾293细胞包装,获得有感染能力的重组腺病毒颗粒。重组腺病毒感染HeLa细胞后,利用Northern blot检测miR-133a成熟分子的表达。结果①成功构建穿梭质粒pAdTrack-miR-133a;②成功构建重组腺病毒质粒pAdeasy-miR-133a;③293细胞包装获得有感染能力的重组腺病毒;④转染的HeLa细胞能够高效表达成熟miR-133a。结论构建了小鼠miR-133a的腺病毒表达载体,证实其转染HeLa后的表达,为后续研究miR-133a的功能打下了基础。

Objective To construct recombinant adenovirus expressing murine muscle-specific microRNA miR-133a and observe its expression in HeLa. Methods The pri-miR-133a genomic sequence was amplified from mouse genomic DNA by PCR and cloned into pAdTrack-CMV plasmid. The shuttle vector was homologously recombined with Adeasy-1 in 13.15183 cells and formed the mammalian expression vector pAdeasy-miR-133a. Furthermore, the recombinant vector was packaged in 293 cells into infectious recombinant adenovirus, which was used to infect HeLa, The expression of mature miR-133a in HeLa was detected by Northern blot. Results We successfully constructed recombinant adenovirus vector that simultaneously expressed miR-133a. The expression of miR-133a was confirmed by Northern blot in infected HeLa. Conclusion The cloning of genomic sequence corresponding to pri-miR-133a and construction of its recombinant adenovirus vector have laid the foundation for further study of miR-133a function.