转化生长因子-β1基因RNAi质粒载体的构建与鉴定  

Construction and identification of plasmid vector of RNA interference of transforming growth factor-β1 gene

作　　者：王翔[1] 胡治平[1] 周为军 汤建光[1] 卢伟[1] 唐湘祁[1] 

机构地区：[1]中南大学湘雅二医院神经内科,湖南长沙410011 [2]湘乡市第一人民医院神经内科,湖南湘乡411400

出　　处：《西安交通大学学报（医学版）》2009年第1期22-26,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.30470602)~~

摘　　要：目的构建人转化生长因子-β1(TGFβ1)基因RNAi质粒载体。方法根据人TGFβ1mRNA序列选择3个靶序列并设计合成相应3对寡核苷酸序列,同时合成1对阴性对照寡核苷酸序列;将以上4对寡核苷酸序列退火后连入pRNA-U6.2/Lenti质粒并分别命名为pRNA-U6.2/Lenti-1、pRNA-U6.2/Lenti-2、pRNA-U6.2/Lenti-3、pRNA-U6.2/Lenti-4。酶切和测序鉴定后,将以上重组质粒转染Hela细胞,运用RT-PCR和ELISA检测TGFβ1mRNA和蛋白的表达水平。结果酶切和测序证实目的寡核苷酸片段已被准确克隆到pRNA-U6.2/Lenti质粒;与对照组相比,pRNA-U6.2/Lenti-1、pRNA-U6.2/Lenti-2转染Hela细胞后,TGFβ1mRNA水平及蛋白水平的表达量均受到明显抑制;其中TGFβ1蛋白水平在pRNA-U6.2/Lenti-1组下降79.2%,在pRNA-U6.2/Lenti-2组下降53.7%,统计有显著性差异(P<0.01)。结论成功构建了人TGFβ1基因RNAi质粒载体,对于TGFβ1高表达疾病的基因治疗奠定了基础。Objective To construct a plasmid vector of RNA interference （RNAi） of transforming growth factor-β1 gene （TGF-β1）, Methods First three target sequences were selected according to TGF-β1 mRNA sequence firstly, then three pairs of oligonucleotide sequences and one pair of negative control oligonucleotide sequence were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pRNA-U6.2/Lenti plasmid, which was then named pRNA-U6. 2/Lenti-1, pRNA-U6. 2/Lenti-2, pRNA-U6. 2/Lenti-3 and pRNA-U6. 2/Lenti-4, respectively. After being identified by restriction enzyme digestion and sequencing, the recombinant plasmids were transfected into Hela ceils. TGF-β1 expression in the transfected ceils was assayed by RT-PCR and ELISA. Results Enzyme digestion and DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNA- U6.2/Lenti plasmid, and TGF-β1 expression in the transfected cells was knocked down significantly by pRNA-U6.2/ Lenti-1 and pRNA-U6.2/Lenti-2 at both the protein and mRNA level compared with those in the control group. TGF-β1 protein was reduced by a percentage of 79.2% and 53.7% in pRNA-U6.2/Lenti-1 and pRNA-U6.2/Lenti-2 group, respectively, which was significantly different from that in the control group （P〈0.01）. Conclusion The RNAi plasmid vector of TGF-β1 was constructed successfully, which laid foundation for gene therapy for diseases with high TGF-β1 expression.

关 键 词：RNAI 转化生长因子Β1 质粒载体 

分 类 号：R246.6[医药卫生—针灸推拿学]