RNA干扰Twist基因真核表达载体的构建与筛选

Construction and screening of plasmid expression vectors encoding short hairpin RNA targeting TWIST gene

目的:构建编码Twist mRNA的短发卡样RNA(shRNA)质粒表达载体,并筛选出基因沉默效果最明显的shRNA质粒表达载体。方法:以Twist mRNA编码区中第777位和第845位序列作为RNA干扰靶点,分别构建2个shRNA质粒表达载体和1个阴性对照质粒表达载体,构建后通过PCR酶切电泳和质粒测序进行鉴定。经鉴定后分别转染稳定表达Twist基因的膀胱癌T24细胞,RT-PCR和Western blotting分别从mRNA和蛋白质水平检测抑制效果。结果:构建的质粒表达载体PCR鉴定均可扩增出400 bp的目的条带,插入片段测序结果与合成的shRNA结果一致。靶向Twist基因的shRNA对膀胱癌T24细胞中Twist基因mRNA抑制率为90%,蛋白质抑制率为86%,两种干扰质粒中shRNA1干扰效果最明显。结论:成功构建了靶向Twist基因的shRNA质粒表达载体,其中抑制效果最为明显的shRNA质粒表达载体为pGenesil-shRNA1质粒。

Objective To construct a plasmid expression vector coding for the short hairpin RNA （shRNA） targeting Twist mRNA. Methods Two plasmid expression vectors coding for shRNA targeting 777 and 845 of Twist gene sequence and a control vector containing random DNA fragment were constructed. The recombinant plasmids were identified by PCR, and then transfected separately into bladder cancer cell line T24. The Twist gene silencing effect was detected by RT-PCR and Western blotting. Results The expected band of 400 bp was amplified from the plasmids coding for shRNA by PCR. By DNA sequencing, it was the same with the insertion element as with the shRNA of synthetic. Transfection of T24 cells expressing Twist gene with the shRNA plasmids resulted in inhibition of Twist mRNA and protein expressions by 900% and 86%, respectively. The shRNA1 had the most obvious effect in Two types of plasmids interference. Conclusion The plasmid expression vectors coding for shRNA targeting Twist mRNA have been constructed successfully, of which pGenesil-shRNA1 most effectively silences Twist gene in T24 cells.