KLF4 siRNA慢病毒载体构建和鉴定

Construction and Identification of siRNA Lentivirus Vector Targeting KLF4 Gene

目的:构建上皮锌指蛋白4(Krüppel-like factor 4,KLF4)siRNA慢病毒载体并进行初步鉴定,为研究KLF4在宫颈细胞癌中的分子机制奠定基础。方法:利用公用网站中提供的RNA干扰序列设计原则,设计4个RNA干扰靶点序列,合成含干扰序列的单链DNA oligo,然后退火配对产生双链,再通过其两端所含酶切位点直接连入酶切后的RNAi慢病毒载体上;将连接产物转入制备好的细菌感受态细胞,PCR鉴定阳性重组子后,送测序验证,测序结果经比对确认正确的克隆,制备编码慢病毒颗粒的重组病毒质粒及其两种辅助包装原件载体质粒,共转染293T细胞,收集富含慢病毒颗粒上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度。收集上清液感染宫颈癌He La细胞,通过q RT-PCR及Western Blot鉴定KLF4 siRNA慢病毒干扰效果。结果:成功构建KLF4 siRNA慢病毒载体。KLF4 siRNA慢病毒感染He La细胞后,q RT-PCR及Western Blot测定结果显示,KLF4表达明显降低。结论:KLF4 siRNA慢病毒载体构建及包装成功,可有效抑制KLF4表达,为研究KLF4生物学功能奠定基础。

Objective： To construct and identify the Lentivirus vector interfering human KLF4 gene in order to further research the function of KLF4 in HeLa cell. Methods： Small interference RNA （siRNA） targetingKLF4 gene was designed and DNA template was synthesized and subcloned into the Lentivirus expression plasmid. The recombinant plasmid KLF4 siRNA was confirmed by PCR and gene sequencing. The KLF4 siRNA and Lentiviral packaging plasmid was transfected into 293T cells by liposome. After collecting and concentrating supematant culture medium to infect HeLa cells. The infected Hela cells with KLF4 siRNA were identified by qRT-PCR and Western blot. Results： The recombinant lentivirus expression plasmidKLF4 siRNA were successfully constructed. The recombinant lentivirus vector KLF4 siRNA could infect HeLa cells successfully. The results of qRT-PCR and Western blot showed that the expression of KLF4 was obviously inhibited in the HeLa cells infected by KLF4 siRNA lentivirus. Conclusions： The recombinant lentivirus vector KLF4 siRNA was successfully constructed which could inhibit the expression of KLF4.