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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。
OBJECTIVE: To design and synthesize three pairs of complementary DNA sequences and one pair of negative control sequences of short hairpin structure by using RNA interference technology and human BIRC5 gene as target gene. Methods: The designed and synthesized single-stranded primer was annealed into double-stranded oligo sequence and ligated into the pMAGic lentiviral plasmid vector linearized by AgeI and EcoR I enzyme digestion. After transformed into DH5α competent cells and screened out positive clones, Positive clones were identified by amplification and DNA sequencing. Results: The PCR product was positive cloned by gel electrophoresis to obtain a 335 bp band, the negative clone was 298 bp. DNA sequencing confirmed that it contained the designed and synthesized sequence. Conclusion: 4 pairs of BIRC5 shRNA recombinant lentiviral expression vector was constructed successfully, which laid the foundation of the research on the effect of BIRC5 siRNA on the proliferation inhibition and induction of apoptosis of tumor cells and gene therapy research.