靶向转酮醇酶样基因1 siRNA表达载体的构建及鉴定

Construction and identification of siRNA expression vectors for silencing transketolase-like gene 1

目的构建针对转酮醇酶样基因1(TKTL1)特异性小干扰RNA(siRNA)真核表达载体并检测其沉默效应。方法采用人U6 SnRNA启动子,分别把被9 bp序列间隔的19 bp长短的TKTL1靶序列的反向重复序列,置于pEGFP-C1-U6质粒载体中,构建成TKTL1短发夹环RNA,产生重组质粒pEGFP-C1-U6/TKTL1,分别用PstⅠ和SalⅠ酶切鉴定及测序分析,并将重组质粒转染人结肠癌细胞株LoVo,通过RT-PCR检测TKTL1基因表达水平的变化。结果酶切鉴定与DNA测序分析证明,TKTL1基因的siRNA表达载体构建正确,RT-PCR结果表明转染重组质粒的LoVo细胞TKTL1基因的表达水平明显降低。结论成功构建了针对TKTL1基因的siRNA表达载体,转染LoVo细胞后可显著抑制TKTL1基因表达。

Objective To construct specific small interfering RNA （siRNA） expression vectors of transketolase-like gene 1 and to detect the silencing effects. Methods A plasmid pEGFP-CI-U6/TKTL1 containing human U6 snRNA promoter was ligated to a 19 bp reverse repeated motif of TKTL1 target sequence with 9 bp spacer. The recombinant pEGFP-C1-U6/TKTL1 plasmids were identified by restriction endonuclease （Psi II and Sal I ） and DNA sequencing, and then transferred into LoVo cell line. The level of TKTLl mRNA was examined by real-time PCR. Results The TKTL1 short hairpin RNA （shRNA） expression frame was successfully inserted into expression vector. Enzyme digestion by Pst I and Sal I showed a 400 bp DNA fragment. The sequence of shRNA recombinant plasmid was the same as that of the designed fragment. Conclusions Expression vectors of shRNA specific for TKTL1 were constructed. The expression of TKTL1 mRNA in LoVo cell line transfected by recombinant pEGFP-C1-U6/TKTL1 plasmids was reduced significantly.