论文部分内容阅读
通过RNAi技术干扰人前列腺癌PC3细胞Bloom(BLM)解旋酶基因的表达。实验前期根据BLM解旋酶基因序列设计并合成两对sh RNA,插入到CMV-cop GFP-T2A-Puro-H1-mcs载体,构建针对BLM解旋酶基因的重组干扰载体CMV-cop GFP-T2A-Puro-H1-mcs-BLM1、CMV-cop GFPT2A-Puro-H1-mcs-BLM2。经测序载体构建成功后,用脂质体将所构建的载体及阴性对照载体转染前列腺癌PC3细胞,通过荧光定量PCR和Western blot检测转染细胞的BLM解旋酶表达情况。检测结果显示,成功构建了BLM解旋酶RNAi真核表达载体;利用脂质体转染PC3细胞后,荧光定量PCR和Western blot鉴定结果表明,BLM解旋酶的表达水平显著降低,即Bloom RNAi真核表达载体在m RNA和蛋白质水平阻断了BLM解旋酶的表达。
Interfere with the expression of Bloom (BLM) helicase gene in human prostate cancer PC3 cells by RNAi technique. Two pairs of sh RNAs were designed and synthesized according to the BLM helicase gene sequence in the early stage of the experiment and inserted into the CMV-cop GFP-T2A-Puro-H1-mcs vector to construct a recombinant interference vector CMV-cop GFP-T2A for the BLM helicase gene -Puro-H1-mcs-BLM1, CMV-cop GFPT2A-Puro-H1-mcs-BLM2. After successful construction of the sequencing vector, the constructed vector and the negative control vector were transfected into prostate cancer PC3 cells by lipofectamine. The expression of BLM helicase in transfected cells was detected by real-time PCR and Western blot. The results showed that the BLM helicase RNAi eukaryotic expression vector was successfully constructed. After transfected into PC3 cells by liposome, the results of quantitative PCR and Western blot showed that the BLM helicase expression was significantly decreased, Eukaryotic expression vectors block the expression of BLM helicase at m RNA and protein levels.