PAK4基因RNAi慢病毒载体的构建与鉴定

目的：构建PAK4基因RNAi慢病毒载体，并对其在涎腺腺样囊性癌细胞株SACC-83细胞中干扰效率进行鉴定。方法：针对PAK4基因RNAi有效靶序列，合成Oligo DNA，退火形成双链DNA，与经AgeⅠ和EcoRⅠ 酶切后的pGCsil-GFP载体连接产生shPAK4i-LV慢病毒载体，PCR筛选阳性克隆，测序鉴定。用shPAK4i-LV载体、pHelper1.0载体和pHelper 2.0质粒共转染包装细胞293T细胞，包装产生慢病毒，以293T细胞绿色荧光蛋白(GFP)的表达水平测定病毒滴度。 结果：PCR扩增出插入片段，测序证实成功构建了PAK4-shRNA慢病毒载体shPAK4i-LV。包装并浓缩慢病毒，滴度为2E+9 TU?mL^-1。将病毒感染SACC-83细胞，real-time PCR检测PAK4的mRNA表达下调70%以上。结论：成功构建了shPAK4i-LV病毒载体并建立了SACC-83-shPAK4i细胞模型，为PAK4在肿瘤信号转导通路中的研究提供工作基础。

Construction and identification of lentiviral vector of RNA interference of PAK4 gene

Objective To construct a lentiviral vector of RNA interference(RNAi) of PAK4 gene and to study the interference efficiency of PAK4 shRNA in SACC-83 cells in vivo.Methods The complementary oligo DNA was designed according to the effective sequence of siRNA targeting PAK4 gene and then synthesized and cloned into the pGCSIL-GFP vector.The obtained lentiviral vector containing PAK4i shRNA was named as shPAK4i-LV,and it was confirmed by PCR and sequencing.The HEK-293T cells were cotransfected with lentiviral vector shPAK4i-LV,pHelper1.0 and pHelper2.0.All virus stocks were produced by LipofectamineTM 2000 transfection.The titer of virus was tested according to the expression level of GFP.Results PCR and DNA sequencing demonstrated that the lentiviral vector shPAK4i-LV of PAK4 shRNA was constructed successfully.The titer of concentrated virus was 2E+9TU·mL-1.The SACC-83 cell line was infected with shPAK4i-LV and the expression of PAK4 gene was inhibited about 70% which was confirmed by real-time PCR.Conclusion The shPAK4i-LV is constructed successfully and SACC-83-shPAK4i transient transfection cell model is established.It provides the basis for research on PAK4 signal transduction pathway in tumor.