论文部分内容阅读
目的:构建携带KIR2DS2基因短发夹状干扰RNA(short hairin RNA,shRNA)的RNAi慢病毒载体。方法:针对已经筛选确定的KIR2DS2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Hpa I和Xho I酶切后的pSicoR-GFP载体[含U6启动子和绿色荧光蛋白(GFP)]连接产生LV-shKIR2DS2慢病毒载体,PCR筛选阳性克隆,测序鉴定。用LV-shKIR2DS2及Packaging system质粒共转染293T细胞,包装产生慢病毒颗粒,以293T细胞GFP蛋白的表达水平测定病毒滴度。结果:PCR和测序证实,成功构建KIR2DS2shRNA的慢病毒载体LV-shKIR2DS2。293T细胞测定病毒滴度为6×108TU/ml。结论:成功构建携带KIR2DS2基因短发夹状干扰RNA的RNAi慢病毒载体。
Objective: To construct RNAi lentiviral vector carrying KIR2DS2 short hairpin RNA (shRNA). Methods: According to the RNAi effective target sequence of KIR2DS2 gene that has been selected and screened, Oligo DNA of the target sequence was synthesized and annealed to form double-stranded DNA. After digested with Hpa I and Xho I, the pSicoR-GFP vector [containing U6 promoter and green fluorescence Protein (GFP)] to generate LV-shKIR2DS2 lentiviral vector, positive clones were screened by PCR and identified by sequencing. 293T cells were cotransfected with LV-shKIR2DS2 and the packaging system plasmids, and the lentiviral particles were packaged to determine the virus titer with the expression level of GFP protein of 293T cells. Results: PCR and sequencing confirmed that the viral titer of LV-shKIR2DS2.293T cells successfully constructed KIR2DS2shRNA was 6 × 108TU / ml. Conclusion: RNAi lentiviral vector carrying KIR2DS2 short hairpin RNA was successfully constructed.