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目的构建Slug基因RNA干扰(RNA interference,RNAi)慢病毒表达载体。方法针对人Slug基因的序列,设计出RNA干扰的靶序列,合成靶序列Oligo DNA,退火形成双链DNA,通过Age I和EcoR I酶切后的pGCSIL-GFP载体连接产生shRNA慢病毒载体,质粒转化感受态细菌,筛选阳性克隆并用插入鉴定引物进行PCR鉴定阳性克隆并测序,同时应用Real-time PCR和Western blot方法检测HCT116结肠癌细胞中Slug基因mRNA和蛋白的表达情况。结果 PCR鉴定与DNA测序证实合成的含Slug shRNA慢病毒载体寡核苷酸链插入正确。Western blot证实Slug RNAi慢病毒载体能够抑制Slug的表达。结论成功构建Slug基因RNAi慢病毒表达载体,为后续感染结肠癌细胞,为探索在结直肠癌发生和发展中的作用奠定基础。
Objective To construct the Slug gene RNA interference (RNAi) lentivirus expression vector. Methods Aiming at the sequence of human Slug gene, target RNA interference was designed. Oligo DNA was synthesized and double-stranded DNA was annealed to generate shRNA lentivirus vector through plasmid pGCSIL-GFP digested with Age I and EcoR I. The plasmid The positive clones were transformed into competent clones. Positive clones were screened by PCR and positive clones were identified by PCR. The expression of Slug mRNA and protein in HCT116 colon cancer cells was detected by Real-time PCR and Western blot. Results PCR identification and DNA sequencing confirmed that the synthetic Slug shRNA lentiviral vector was inserted correctly. Western blot confirmed Slug RNAi lentiviral vector can inhibit Slug expression. Conclusion The RNAi lentiviral vector of Slug gene was successfully constructed, which could be used as a follow-up vector for colon cancer cells in order to explore the role of Slug RNAi in the development and progression of colorectal cancer.