| 摘要: |
| 目的 构建PDK1基因短发夹RNA(Short hairpin RNA, ShRNA)慢病毒载体（Lentiviral vector, LV），鉴定其对PC-3人前列腺癌细胞PDK1基因的干扰效应。方法 GeneBank检索PDK1基因序列，参考该序列设计PDK1特异性过干扰序列，分别酶切过表达用载体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。将构建的慢病毒载体感染PC-3人前列腺癌细胞，采用Real-time PCR鉴定PDK1基因干扰效应。结果 测序证实成功构建PDK1基因RNA干扰（RNA interference, RNAi）慢病毒载体，病毒滴度为: LV3- PDK1-RNAi 1×108TU/mL。与空白对照组（Blank）和阴性对照组（sh-NC）相比，LV-PDK1-RNAi组PDK1-mRNA转录水平显著下降（P<0.001）。结论 成功构建PDK1基因RNA干扰慢病毒载体，获得稳定沉默PDK1基因的PC-3人前列腺癌细胞株，为后续研究PDK1在前列腺癌细胞模型中的作用奠定基础。 |
| 关键词: RNA干扰 PDK1 转染 前列腺癌 慢病毒载体 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学(编号: 81460387) |
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| Construction and identification of short hairpin RNA plasmids targeting human PDK1 gene and its silencing effect in PC-3 cell |
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Hao Nan1,2,3
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1.the People'2.'3.s Hospital of Guangxi Zhuang Autonomous Region
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| Abstract: |
| Objective The aim of this study was to construct RNA interference (RNAi) lentiviral vector particles targeting the human PDK1 gene. Methods A short hairpin RNA (shRNA) targeting the human PDK1 gene was designed, synthesized and transfected into 293T cells. Identify the shRNA sequence exhibiting the inhibition efficiency; based on this, recombinant lentiviral plasmids were constructed and co?transfected into 293T cells with packaging plasmids for the production of lentiviral particles. Results The virus titer was calculated to be 1×108TU/mL. qRT-PCR results showed that the PDK1 mRNA expression level of the LV3-PDK1-shRNA group was significantly lower than the negative control group and the blank group, with the inhibition rate of 88%. Conclusions RNAi lentiviral vector particles targeting the human PDK1 gene was successfully obtained in the present study, which laid the foundation for the further research on the role of PDK1 in prostate cancer cell models. |
| Key words: RNA interference PDK1 Transfection Prostate cancer Lentiviral vector |
