摘要
目的 设计和构建EOLA1基因特异性的小干扰RNA质粒 ,并初步验证其对靶基因的抑制作用。方法 将靶点特异性的寡核苷酸连接到经ApaⅠ EcoRⅠ酶切线性化的pBS/U6质粒 ,测序验证 ,阳性重组质粒转染ECV3 0 4细胞 ,半定量RT PCR方法检测靶基因的表达 ,阳性重组质粒与pEGFP EOLA1共转染 ,观察外源报告基因绿色荧光蛋白的表达。结果 构建的阳性重组质粒转染能够抑制靶基因EOLA1mRNA的表达 ,并能够显著抑制EOLA1 EGFP融合蛋白的表达。结论 成功构建了针对EOLA1基因的siRNA质粒 ,为进一步研究该基因的功能奠定了基础。
Objective To design and construct U6 promoter-based small interfering RNA (siRNA) expression plasmids and explore their effects on the expression of endothelium overexpression lipopolysaccharide associated factor 1 (EOLA1). Methods DNA oligonucleotides targeting EOLA1 at different locations were synthesized and inserted into ApaⅠEcoRⅠ linearized pBS/U6 plasmid. The inserted sequences were verified by DNA sequencing. ECV304 cells were transfected by positive reconstructs and the expression of EOLA1 mRNA was detected by semi-quantitive RT-PCR. The expression of EGFP-EOLA1 fusion protein was observed after cells were co-transfected by pEGFP-EOLA1 and siRNA reconstructs. Results The plasmid-based RNAi approach could specially knock down EOLA1 gene expression. Conclusion Small interference RNA expression plasmids have been constructed successfully, which paves the way for future studies of EOLA1 gene.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2005年第3期200-202,共3页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目 ( 30 2 70 6 72 )
国家自然科学基金资助重点项目 ( 10 332 0 6 0 )~~
