摘要
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 and screen specific artificial microRNA （amiRNA） expression plasmids targeting porcine Toll-like receptor 7 （TLRT） gene. Methods The 1984 -2649 bp sequence of the porcine TLR7 cDNA was amplified by RT-PCR and inserted into plasmid pEGFP-N1 to construct the fusion expression vector pTLR7-EGFP. Five amiRNAs targeting porcine TLR7 gene were designed and cloned into pcDNA5-miR to construct the recombinant interfering plasmids pcDNA5-miRTLRT. NIH-3T3 cells were co-transfected with plasmids pTLRT-EGFP and pcDNAS-miRTLRT. The expression levels of amiTLR7 were monitored by RT-PCR and their silencing efficiencies were detected by fluorescent microscopy and flow cytometry. Results All five amiTLRTs were successfully constructed and could effectively silence the expression of TLR7 gene in NIH-3T3 cells with the inhibition efficiencies ranging from 36.99% to 97.28%, among which amiTLR7-3 had the best interference efficiency. Conclusion The specific artificial amiRNA expression plasmids targeting porcine TLR7 gene have been successfully constructed, and the optimal amiTLR？ with the highest inhibition efficiency has been screened.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第1期18-21,26,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(31001052)
教育部创新团队基金(IRT0978)
江苏省优势学科建设工程资助项目(2010年)
