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目的:构建介导大鼠Ppif基因沉默的慢病毒载体转移质粒pGCL-Ppif,为进一步包装慢病毒载体奠定基础。方法:以大鼠Ppif基因为靶基因,根据RNA干扰(RNAi)序列设计原则,设计4对有小发夹结构的RNAi靶点序列,退火形成双链DNA,双酶切后定向克隆到慢病毒载体转移质粒pGCL-Ppif中,构建4个含靶基因片段的重组慢病毒载体转移质粒pGCL-Ppif,并对质粒进行PCR及测序鉴定。结果:Ppif的短发夹RNA(shRNA)片段被成功克隆到慢病毒载体转移质粒pGCL-GFP中,4对插入序列与设计的靶基因片段完全一致。结论:构建了能够表达4个含Ppif靶基因片段的慢病毒载体转移质粒,为进一步包装介导Ppif基因沉默的慢病毒载体奠定了基础。
OBJECTIVE: To construct a lentiviral plasmid pGCL-Ppif that mediates the silencing of Ppif gene in rats, which lays a foundation for further packaging of lentiviral vector. Methods: According to the design principle of RNA interference (RNAi) sequence, four pairs of RNAi target sequences with small hairpin structure were designed and annealed to form double-stranded DNA. The target gene was cloned into lentivirus In the vector pGCL-Ppif, four recombinant lentiviral vector containing the target gene fragment was constructed to transfer the plasmid pGCL-Ppif, and the plasmid was identified by PCR and sequencing. Results: The short hairpin RNA (shRNA) fragment of Ppif was successfully cloned into the lentiviral vector transfer plasmid pGCL-GFP. The four pairs of insertions were identical with the designed target gene fragment. CONCLUSION: Construction of lentiviral vector transfer plasmid capable of expressing 4 Ppif target gene fragments lays the foundation for further packaging lentiviral vectors that mediate silencing of Ppif gene.