FAK基因RNAi慢病毒载体的构建与鉴定
Construction and Identification of FAK RNAi Lentiviral Vector
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摘要: 目的 构建FAK基因RNA干扰慢病毒载体。方法 选定FAK基因RNAi靶序列，合成靶序列的Oligo DNA，退火形成双链DNA（引物），与经XhoⅠ和HpaⅠ双酶切后的慢病毒载体pLentilox3.7（pLL3.7）连接，产生pLL3.7 FAK慢病毒载体， 经XbaⅠ和NotⅠ酶切电泳筛选阳性克隆，测序鉴定。用PHR、pVSVG和pLL3.7 FAK慢病毒载体共转染包装细胞293T细胞，包装产生慢病毒，以293T细胞GFP蛋白的表达水平测定病毒滴度。结果 酶切和测序证实，构建出了FAK shRNA的慢病毒载体pLL3.7 FAK。包装慢病毒，测得离心的浓缩病毒悬液的滴度为4×107TU/ml。结论 成功构建FAK基因RNAi慢病毒载体，为肿瘤细胞生物学行为及功能研究提供了一个有用的工具。Abstract: Abstract:Objective To construct a lentiviral RNA interfered vector of FAK gene. Methods The target sequence of FAK gene which can be effectively silenced with RNA inference was confirmed.The cDNA containing both sense and antisense Oligo DNA fragments of target sequence was designed，and cloned into the pLentilox3.7(pLL3.7) vector which was digested by XhoⅠ and HpaⅠ.The obtained lentiviral vector containing FAK shRNA was confirmed by digestion and sequencing.Using lentiviral vector PHR 、pVSVG and pLL3.7 FAK to cotransfect 293T cells，then the lentivirus were collected.The titer of virus was tested according to the expression level of GFP. Results Digestion and DNA sequencing demonstrated that lentivirus vector pLL3.7 FAK was constructed successfully which can produce FAK shRNA.The titer of concentrated virus was 4×107TU/ml. Conclusion The lentivirus RNAi vector targeting FAK was constructed successfully.It provides a useful tool for the study of biological behavior and function of tumor cells.
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