Rac1基NRNAi慢病毒载体的构建与鉴定

背景：Rac1是通过信号转导来调节癌细胞的侵袭、增殖，它在各种肿瘤中都有表达。目的：构建RAC1基因RNA干扰慢病毒载体，并检测其干扰效率。方法：应用Western blot检测Rac1基因在舌癌细胞中的表达。针对筛选确定的Rac1基因RNAi有效靶序列，合成靶序列的Oligo DNA，退火形成双链DNA，与经AgeI和EcoRI双酶切后的pMagic4.1载体连接产生短发卡RNA慢病毒载体，PCR筛选阳性克隆，测序鉴定后将质粒分别和包装质粒混合物共转染293T细胞，采用Real-Time PCR筛靶，将筛靶成功的质粒进行慢病毒包装，再感染Tca8113细胞。荧光定量PCR和Western blot检测鉴定Rac1基因表达的干扰效果以确定其生物活性。结果与结论：成功构建了RAC1干扰载体，Western blot检测显著抑制Rac1蛋白表达，经Real-time PCR检测pLVT447的抑制率达70%。成功的构建了Rac1基因RNAi慢病毒载体，为RNAi用于靶向Rac1的基因治疗舌癌（Tca8113）提供了有效的siRNA靶序列。

Construction and identification of RNA interference lentiviral vector for Racl gene

BACKGROUND： Rac1, which is expressed in a variety of tumors, regulates the invasion and proliferation of cancer cells through signal transduction. OBJECTIVE： To construct a RNA interference lentiviral vector that is capable to knock down Rac1 gene, and to detect its interference efficiency. METHODS： The gene expression of Rac1 in tongue cancer cells was detected by western blot. The Oligo DNA containing target sequence was synthesized according to the previously confirmed effective sequence of small interfering RNA targeting Rac1 gene. Double-stranded DNA was constructed after annealing, and was cloned into the pMagic4.1 vector after digest with AgeI/EcoRI to construct a lentiviral vector which express short hairpin RNA. Positive clones were identified using PCR. The plasmids and packaging plasmids were cotransfected into 293T cells. The small interfering RNA expression cassettes were generated by real-time PCR; the successfully constructed plasmids were packaged by lentivirus and then transfected into Tca8113 cell. The interference effect of Rac1 gene expression was assayed by fluorescence quantitative PCR and western blot to explore its biological activity. RESULTS AND CONCLUSION： RAC1 interference vector was constructed successfully. Western blot results showed that the protein expression of Rac1 was decreased significantly; Real-time PCR results showed that the inhibition rate of pLVT447 was up to 70%. A lentivirus-mediated RNAi vector containing Rac1 gene was successfully constructed, which provides effective small interfering RNA target sequence for the application of RNA interference in the targeting gene therapy of tongue cancer using Rac1.