| 摘要: |
| ［摘要］ 目的 构建PDK1基因短发夹RNA(shRNA)慢病毒载体，鉴定其对PC-3细胞PDK1基因的干扰效应。方法 在GenBank数据库检索PDK1基因序列，参考该序列设计PDK1特异性过干扰序列，酶切过表达用载体LV3，纯化酶切产物后进行定向连接。将重组干扰病毒质粒与慢病毒包装质粒共转染293T细胞，测定病毒滴度。将构建的慢病毒感染PC-3细胞，采用real-time RT-PCR法鉴定其PDK1基因干扰效应。结果 测序结果证实成功构建PDK1基因RNA干扰（RNAi）慢病毒载体，慢病毒滴度为1×108 TU/ml。real-time RT-PCR结果显示，LV3-PDK1-shRNA慢病毒感染PC-3细胞可显著抑制其PDK1 mRNA的表达（P<0.05）。结论 成功构建PDK1基因RNAi慢病毒载体，获得稳定沉默PDK1基因的PC-3细胞株，为后续研究PDK1基因在前列腺癌细胞模型中的作用奠定基础。 |
| 关键词: RNA干扰 3-磷脂酰肌醇依赖的蛋白激酶-1 转染 前列腺癌 慢病毒载体 |
| DOI：10.3969/j.issn.1674-3806.2021.08.08 |
| 分类号:R 34 |
| 基金项目:国家自然科学基金项目（编号：81460387） |
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| Construction of PDK1 gene shRNA lentiviral vector and identification of its interference effect |
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HAO Nan, NONG De-yong, LI Zuan, et al.
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Department of Urology, the People′s Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China
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| Abstract: |
| ［Abstract］ Objective To construct the 3-phosphoinositide dependent protein kinase-1(PDK1) gene short hairpin ribonucleic acid(RNA)(shRNA) lentiviral vector, and to identify its interference effect on the PDK1 gene of prostate cancer cells(PC-3). Methods The PDK1 gene sequence was searched in GenBank database, and the PDK1 specific over-interference sequence was designed according to the sequence. The LV3 vector was linearized for overexpression using restriction enzymes, and the linearized products were purified and the directed ligation was conducted. The recombinant interfering virus plasmid and the lentivirus packaging plasmid were co-transfected into 293T cells, and the virus titer was determined. The constructed lentivirus was infected with PC-3 cells, and the PDK1 gene interference effect was identified by real-time reverse transcription polymerase chain reaction(real-time RT-PCR) method. Results The sequencing results confirmed that the PDK1 gene RNA interference(RNAi) lentiviral vector was successfully constructed, and the lentiviral titer was 1×108 TU/ml. The results of real-time RT-PCR showed that LV3-PDK1-shRNA lentivirus infection of PC-3 cells could significantly inhibit the expression of PDK1 mRNA in PC-3 cells(P<0.05). Conclusion The PDK1 gene RNAi lentiviral vector is successfully constructed, and the PC-3 cell line stably silencing the PDK1 gene is obtained, which lays a foundation for the future studies of the role of PDK1 gene in prostate cancer cell models. |
| Key words: Ribonucleic acid(RNA) interference(RNAi) 3-Phosphoinositide dependent protein kinase-1(PDK1) Transfection Prostate cancer Lentiviral vector |
