Bcl-2shRNA慢病毒表达载体的构建与鉴定

Construction and identification of Bcl-2 shRNA lentiviral expression vector

目的:应用分子克隆技术,针对人bcl-2目的基因设计3个siRNA靶点和一个阴性对照序列,构建4对shRNA重组慢病毒表达载体,并进行测序验证鉴定。方法:以设计的有效靶序列进行引物合成,将单链引物退火成双链oligo序列,连接入经Age I和EcoR I双酶切线性化的慢病毒RNA干扰载体中,经转化DH5α感受态细胞并筛选出阳性转化子,采用PCR扩增和DNA测序分别鉴定阳性克隆。结果:PCR扩增产物经凝胶电泳后阳性克隆得到335bp条带,阴性克隆得到298 bp条带,DNA测序结果证实其含有设计合成序列。结论:成功构建4对bcl-2shRNA重组慢病毒表达载体,为研究靶向bcl-2 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。

Objective：Design and synthesis complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence with RNA interference and human Bcl-2 gene as target gene,to construct and identify a lentiviral interference vector.Methods：The designed and synthesised Single-Stranded primer were annealed to Double-Stranded oligo sequences and subcloned into linear 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：335bp straps of positive clone and 298bp 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：4 pairs of Bcl-2 shRNA recombinant lentiviral expression vector are constructed successfully,which laid foundation for research of Bcl-2 siRNA target to tumor cells proliferation inhibition,induction apoptosis and gene therapy.