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目的:设计并构建CDK1-shRNA质粒表达载体,转染肝癌HepG2细胞,验证shRNA对肝癌HepG2细胞CDK1基因的沉默效应,便于进一步研究CDK1的功能。方法:设计针对CDK1mRNA靶序列的小发夹状RNA,化学合成含茎环结构的正义链与反义链,退火形成带内切酶粘/平末端的双链后,与酶切后的 pSIREN-RetroQ-ZsGreen载体片段进行连接、转化。经测序等方法鉴定重组克隆。将重组载体质粒经脂质体包裹转染肝癌HepG2细胞,realtime PCR及Western blot检测RNA干扰效果。结果:重组克隆经测序证实插入的序列正确,realtime PCR及Western blot检测证实设计的3条RNA干扰序列有效沉默了肝癌HepG2细胞中的内源性CDK1。结论:所制备的CDK1-shRNA在肝癌HepG2细胞中可获得高效转染,并能产生特异性的基因沉默效应,以CDK1为靶向的shRNA能够有效下调CDK1基因的表达,对周期依赖激酶CDK1功能的研究奠定基础。
OBJECTIVE: To design and construct CDK1-shRNA plasmid expression vector and transfect HepG2 cells to verify the silencing effect of shRNA on CDK1 gene in HepG2 hepatocellular carcinoma so as to further study the function of CDK1. METHODS: Small hairpin RNA targeting CDK1 mRNA was designed to chemically synthesize the sense and antisense strands containing the stem-loop structure and annealed to form the double-stranded end-only double-stranded strand. The digested pSIREN- RetroQ-ZsGreen vector fragments were ligated and transformed. The recombinant clones were identified by sequencing and other methods. The recombinant plasmids were transfected into HepG2 cells by lipofectamine. Realtime PCR and Western blot were used to detect the effect of RNA interference. Results: The inserted sequence was confirmed by sequencing. Realtime PCR and Western blot confirmed that three designed RNA interference sequences effectively silenced endogenous CDK1 in HepG2 cells. Conclusion: The prepared CDK1-shRNA can be efficiently transfected into HepG2 hepatocarcinoma cells and produce specific gene silencing effect. The shRNA targeted by CDK1 can effectively down-regulate the expression of CDK1 gene and inhibit the expression of cyclin dependent kinase CDK1 The research laid the foundation.