靶向白介素-1α基因沉默的Hela229稳定细胞系的构建  

Establishment of Hela229 Stable Cell Lines with Interleukin-1 Alpha Gene Knockdown

作　　者：张宁洁[1] 张丽娜[1] 李伟[1] 林琳[1] 高劲松[2] 程文[1] 余平[1] 

机构地区：[1]中南大学基础医学院免疫系,湖南长沙410008 [2]长治医学院基础部免疫学教研室,山西长治046000

出　　处：《激光生物学报》2013年第5期441-446,440,共7页Acta Laser Biology Sinica

基　　金：国家自然科学基金资助项目(81071403)

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。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-1 α of Hela229 cells.Successfully established the Hela229 stable cell line with IL-1α gene knockdown.

关 键 词：IL-1Α HELA229细胞 SHRNA 

分 类 号：Q78[生物学—分子生物学]