EGF和IGF-1受体特异性siRNA真核表达载体的设计、构建及鉴定

目的构建能表达靶向EGF RmRNA和IGF-1 RmRNA的siRNA真核表达质粒。方法从GeneBank中搜索EGFR（NM 000875）和IGF-1 R（NM 201283）基因组标准序列,根椐siRNA设计原理各基因选取两段19bp的碱基序列作为靶目标,并根据其序列构建真核表达质粒。随机选取一段与人类基因无同源性的碱基序列,构建表达siRNA的真核表达质粒作为质粒对照psiPC。转染后分别培养12h、24h、36h、48h、60h、72h,以转染率高的时间点的鼻咽癌细胞做流式细胞仪检测转染率,并采用共聚焦显微镜检测质粒绿色荧光蛋白的表达。结果质粒psiEGFR1、psiEGFR2、psiIGF-1 R1、psiIGF-1 R2和psiPC酶切后均可见400bp小带,与预期插入的目的基因大小一致,基因测序证实碱基序列与模板序列相符;经荧光显微镜下摄片计算转染效率发现：在转染48h达最高（55%～60%之间）;流式细胞仪检测转染后48h的转染率为57.45%,共聚焦显微镜下观察均见大量的细胞表达绿色荧光。结论本实验构建了靶向EGFRmRNA和IGF-1 RmRNA、表达短发夹RNA（shRNA）的真核表达质粒。重组质粒能有效转染人鼻咽癌细胞并在其中表达,能下调鼻咽癌细胞EGFR和IGF-1 R的表达。

Design,Construction and Identification of siRNA Eukaryotic Expression Vectors Targeting to EGF and IGF-1 Receptors

Objective To design,construct and identify siRNA expression vectors.targeting to EGF and IGF-1 receptors.Methods The primary structures of EGFR and IGF-1 R cDNA were found in GeneBank,respectively.Then the structure analysis was done according to the strategy of RNAi,which determined the specific sequences to design siRNA plasmid.Two sequences of EGFR（psiEGFR1 and psiEGFR2） and IGF-1 R（psiIGF-1 R1 and psiIGF-1 R2）,involved in fluorescein gene were synthesized based on the specific base sequence.Plasmid control psiPC-a random sequence was also constructed.Cell line CNE2 was treated daily with these different vectors.The psiEGFR1,psiEGFR2,psiIGF-1 R1 and psiIGF-1 R2 were identified by gene base sequencing and were identified by electrophoresis.After administration of psiEGFR1,psiEGFR2,psiIGF-1 R1 and psiIGF-1 R2,fluorescence expression was detected by confocal microscopy.EGFR and IGF-1 R protein expression of the transfected cells were determined by S-P immunohistochemistry.Results There was a 400bp balteum in psiEGFR1,psiEGFR2,psiIGF-1 R1,psiIGF-1 R2 and psiPC after cut by SalI,which was identical with the size of the objective gene.The gene sequence of psiEGFR1,psiEGFR2,psiIGF-1 R1 and psiIGF-1 R2 was identical with the anticipation.Many cells gave out green fluorescent and EGFR and IGF-1 R expression in CNE2 significantly was decreased after treated by psiEGFR1,psiEGFR2,psiIGF-1 R1,psiIGF-1 R2.Conclusion siRNA targeting to expression vectors have been succeeded.All of these new vectors can be effectively transfected into CNE2 cells and are expressed in CNE2.