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目的:构建KCTD10干扰RNA慢病毒载体。方法:合成KCTD10干扰序列,构建GV248-KCTD10-RNAi慢病毒干扰载体,利用PCR与测序验证阳性克隆。将KCTD10 siRNA干扰病毒载体与质粒pHelper1.0和质粒pHelper2.0三个载体共同转染293T细胞,使细胞合成并分泌释放病毒颗粒。收集病毒颗粒并检测病毒滴度。结果:GV248-KCTD10-RNAi慢病毒载体成功被连接转化,测序结果与设计序列一致;共转后收获KCTD10 siRNA慢病毒颗粒,检测其滴度为5×10~8TU/mL。结论:成功包装了KCTD10 siRNA慢病毒颗粒,为后续进一步研究KCTD10基因在糖尿病视网膜病变中的作用奠定了基础。
Objective: To construct lentiviral vector of KCTD10 interfering RNA. Methods: The KCTD10 interference sequence was synthesized and the GV248-KCTD10-RNAi lentiviral vector was constructed. The positive clones were verified by PCR and sequencing. 293T cells were co-transfected with KCTD10 siRNA interference virus vector and three vectors of plasmid pHelper1.0 and plasmid pHelper2.0 to synthesize and release the virus particles. Virus particles were collected and virus titers were detected. Results: The GV248-KCTD10-RNAi lentiviral vector was ligated and transformed successfully. The sequencing result was in accordance with the designed sequence. After co-transfection, the KCTD10 siRNA lentivirus particles were harvested and the titer was 5 × 10-8 TU / mL. Conclusion: The successful packaging of KCTD10 siRNA lentiviral particles lays the foundation for further study on the role of KCTD10 gene in diabetic retinopathy.