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目的构建猪Toll样受体7(TLR7)基因的人工miRNA(amiRNA)特异性表达载体,筛选有效amiRNA。方法 RT-PCR扩增猪TLR7基因的1984～2648 bp序列,构建融合表达载体pTLR7-EGFP。设计针对猪TLR7的5对amiRNA,构建重组干扰质粒pcDNA5-miRTLR7。将pcDNA5-miRTLR7和pTLR7-EGFP共转染NIH-3T3细胞,通过RT-PCR检测amiTLR7的表达,以荧光显微镜观察和流式细胞术分析干扰效率。结果 5个amiTLR7都成功表达,且均能沉默NIH-3T3细胞中TLR7基因的表达,抑制效率为36.99%到97.28%,其中amiTLR7-3效果最佳。结论成功构建了针对猪TLR7基因的amiRNA表达质粒,筛选出了沉默猪TLR7基因的最佳干扰序列。
Objective To construct artificial amiRNA (amiRNA) specific expression vector of porcine Toll - like receptor 7 (TLR7) gene and screen for effective amiRNA. Methods The 1984 ~ 2648 bp sequence of porcine TLR7 gene was amplified by RT-PCR and the fusion expression vector pTLR7-EGFP was constructed. Five pairs of amiRNA targeting pig TLR7 were designed to construct recombinant interference plasmid pcDNA5-miRTLR7. The NIH-3T3 cells were co-transfected with pcDNA5-miRTLR7 and pTLR7-EGFP, and the expression of amiTLR7 was detected by RT-PCR. The interference efficiency was analyzed by fluorescence microscopy and flow cytometry. Results All of the five amiTLR7 cells were successfully expressed, and all of them were able to silence the expression of TLR7 gene in NIH-3T3 cells. The inhibitory efficiency was 36.99% to 97.28%, of which amiTLR7-3 was the best. Conclusion The amiRNA expression plasmid targeting TLR7 gene was successfully constructed and the best interference sequence of TLR7 gene was screened out.