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目的:构建针对人BRMS1基因的RNA干扰表达载体并检测其有效性,为后续研究提供基础。方法:设计合成针对人BRMS1的特异性si RNA前体寡核苷酸,依次通过退火,连接,构建含前体si RNA的重组质粒pcDNA6.2-GW/EmGFP-BRMS1-si-RNA。经测序鉴定后,通过脂质体介导将其与pDsRed2-N1-BRMS1质粒(包含BRMS1红色荧光融合蛋白的载体)共转染人胚肾293细胞。分别用倒置荧光显微镜和流式细胞术观察转染细胞中红色荧光蛋白的强度,检测干扰效果。结果:确定了两对针对人BRMS1的si RNA片段。经测序鉴定,两种含前体si RNA的质粒均构建成功。共转染后,倒置荧光显微镜下观察可见,两种si R-NA均能明显降低细胞中BRMS1的表达;用流式细胞仪检测证实,si RNA1及si RNA2分别使BRMS1表达下降67.33%和76.67%,与阴性对照组比较差异有统计学意义,P<0.05。结论:成功构建了针对人BRMS1基因的si RNA表达质粒,为进一步利用RNA干扰技术研究BRMS1功能与作用机制奠定了基础。
OBJECTIVE: To construct RNA interference expression vector targeting human BRMS1 gene and test its effectiveness, providing the basis for further study. Methods: The specific si RNA precursor oligonucleotide targeting human BRMS1 was designed and synthesized. The recombinant plasmid pcDNA6.2-GW / EmGFP-BRMS1-si-RNA containing precursor si RNA was constructed by annealing and ligating in turn. After sequencing, human embryonic kidney 293 cells were co-transfected with pDsRed2-N1-BRMS1 plasmid (vector containing BRMS1 red fluorescent fusion protein) by liposome. The intensity of the red fluorescent protein in the transfected cells was observed by inverted fluorescence microscope and flow cytometry respectively, and the interference effect was detected. Results: Two pairs of si RNA fragments against human BRMS1 were identified. After sequencing, two plasmids containing precursor si RNA were successfully constructed. After co-transfection, the results of inverted fluorescence microscope showed that both si R-NA significantly reduced the expression of BRMS1 in the cells. Flow cytometry confirmed that si RNA1 and si RNA2 decreased the expression of BRMS1 by 67.33% and 76.67, respectively %, Compared with the negative control group, the difference was statistically significant, P <0.05. CONCLUSION: The si RNA expression plasmid targeting human BRMS1 gene has been successfully constructed, which lays the foundation for the further study on the function and mechanism of BRMS1 using RNA interference technology.