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目的:构建能高效表达成熟miR-21小分子的腺病毒载体,探讨其对TLR4基因的靶向调控关系。方法:以正常小鼠基因组DNA为模板,PCR扩增pri-miR-21基因,克隆至穿梭载体pAdTrack-CMV中经PCR、酶切及基因测序分析正确无误后,与pAdEasy-1腺病毒骨架质粒进行同源重组,并利用293A细胞,包装成pri-miR-21重组腺病毒感染HeLa细胞,通过Western blot检测TLR4的蛋白表达水平,验证miR-21与TLR4靶向调控的关系。结果:经PCR、酶切、测序及GFP表达证实,成功构建携带pri-miR-21基因的重组腺病毒载体并制备出高滴度重组病毒。Western blot证实,miR-21可抑制TLR4蛋白的表达。结论:所制备的小鼠pri-miR-21重组腺病毒,可以高效表达成熟miR-21小分子,能够抑制靶基因TLR4的表达。
OBJECTIVE: To construct an adenovirus vector capable of efficiently expressing mature miR-21 small molecule, and to investigate its regulation of TLR4 gene targeting. Methods: The normal mouse genomic DNA was used as a template to amplify the pri-miR-21 gene by PCR and cloned into the shuttle vector pAdTrack-CMV. After PCR, restriction analysis and gene sequencing analysis, HeLa cells were infected with 293A cells and packaged into pri-miR-21 recombinant adenovirus. The protein expression of TLR4 was detected by Western blot to verify the relationship between miR-21 and TLR4. Results: The recombinant adenovirus carrying pri-miR-21 gene was successfully constructed and confirmed by PCR, restriction enzyme digestion, sequencing and GFP expression. Western blot confirmed that miR-21 can inhibit TLR4 protein expression. Conclusion: The recombinant mouse pri-miR-21 recombinant adenovirus can efficiently express mature miR-21 and inhibit the expression of target gene TLR4.