BIRC5 shRNA慢病毒表达载体的构建与鉴定

Construction and identification of BIRC5 shRNA lentiviral expression vector

目的:应用RNA干扰技术,以人BIRC5基因为靶基因,设计合成3对短发夹结构的互补DNA序列和一对阴性对照序列,构建慢病毒干扰载体并鉴定。方法:将设计合成的单链引物退火成双链oligo序列,连接入经AgeI和EcoR I双酶切线性化的pMAGic慢病毒质粒载体中,经转化DH5α感受态细胞并筛选出阳性克隆,采用PCR扩增和DNA测序鉴定阳性克隆。结果:PCR扩增产物经凝胶电泳阳性克隆得到335 bp条带,阴性克隆得到298 bp条带,DNA测序结果证实其含设计合成序列。结论:4对BIRC5 shRNA重组慢病毒表达载体构建成功,为研究靶向BIRC5 siRNA对肿瘤细胞增殖抑制与诱导凋亡作用及基因治疗研究奠定了实验基础。

AIM：Design and synthesis complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence by RNA interference technique and construct and identify a lentiviral interference vector with human BIRC5 gene as target gene.METHODS：The designed and synthesised Single-Stranded primer were annealed to Double-Stranded oligo sequences and subcloned into linear pMAGic lentiviral plasmid vector digested by enzyme Age I and EcoR I.Screening positive clone after transformed into DH5α competent cells and identified by PCR amplification and DNA sequencing.RESULTS：335 bp straps of positive clone and 298 bp straps of negative clone form PCR amplification production have been obtained after gel electrophoresis,the designed and synthesised sequences have been contained in these clone straps confirmed by the result of DNA sequencing.CONCLUSION：Four pairs of BIRC5 shRNA recombinant lentiviral expression vector were constructed successfully,which laid the foundation for researching the inhibition of BIRC5 siRNA target against tumor cells proliferation,induction apoptosis and gene therapy.