【摘要】 目的:构建针对IL-1α基因的shRNA表达载体,筛选能够抑制Hela229细胞内源性IL-1α表达的shRNA,建立无内源性IL-1α表达的Hela229稳定细胞系。方法:根据shRNA的设计原则,以IL-1αcDNA oligo为模板设计一段21 bp核苷酸目标序列,构建成siRNA的DNA模板并克隆到shRNA表达载体pRNAT-U6.1/Neo中,获得靶向抑制IL-1α基因的重组shRNA质粒,转染Hela229细胞,经G418筛选后获得单克隆稳定细胞株,用ELISA方法在蛋白水平上检测IL-1α基因的沉默效果。结果:经酶切鉴定和测序分析确定IL-1α-shRNA重组质粒构建正确,ELISA筛选出能够显著抑制内源性IL-1α表达的shRNA,获得沉默内源性IL-1α表达的单克隆稳定的Hela229细胞株。结论:靶向IL-1α基因的重组shRNA表达质粒可显著抑制Hela229细胞内源性IL-1α的表达,成功构建靶向IL-1α基因沉默的Hela229稳定细胞系。更多还原

【Abstract】 Objective: Constructing IL-1α gene targeted short hairpin RNA interference expression vector,which can inhibit the endogenous expression of IL-1α of Hela229 cells,to establish the endogenous IL-1α gene knockdown stable cell line. Method: Design a 21 bp target nucleotide sequence according to the principle of shRNA desigation,clone the DNA template of siRNA into the shRNA expression vector pRNAT U6. 1 / Neo,gain the inhibition of IL-1α gene recombinant shRNA expression plasmid,transfect the plasmid into Hela229 cells. Monoclonal stable cell lines was established after screening by G418. Detect the IL-1α supression effect at the protein level via the method of ELISA. Results: IL-1α-shRNA carrier recombination were determined the correct construction through the enzyme identification and sequencing analysis. ELISA results showed that the restructuring of the IL-1α-shRNA expression vector can significantly inhibit the expression of endogenous IL-1α of Hela229 cells. Conclusion: Targeted IL-1α gene recombinant shRNA expression plasmid can significantly inhibit the expression of endogenous IL-1α of Hela229 cells. Successfully established the Hela229 stable cell line with IL-1α gene knockdown.更多还原