靶向人VEGF165的SiRNA慢病毒的构建和鉴定(1)
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[摘要]目的：构建靶向VEGF基因的SiRNA慢病毒载体，并包装病毒。 方法：针对已经筛选确定的SiRNA 有效靶序列，合成靶序列的OligoDNA，经双酶切后与PGC SIL-GFP慢病毒载体连接，构建慢病毒载体PGCSIL-SiVEGF，以293T细胞包装得到干扰病毒LV-SiVEGF，根据细胞表达绿色荧光蛋白水平计算病毒滴度；病毒感染人脐静脉内皮细胞HUVEC，通过定量RT-PCR检测VEGF的mRNA表达。结果：测序结果表明，靶向人VEGF的SiRNA慢病毒载体构建成功，包装后的慢病毒滴度为2×109 ifu/μl；感染HUVEC细胞后，实验组和对照组相比VEGF mRNA表达水平显著降低。结论：成功构建人VEGF SiRNA 慢病毒LV-SiVEGF。
[关键词]VEGF；慢病毒载体；RNA干扰技术
[中图分类号]Q813.1[文献标识码]A[文章编号]1008-6455(2011)07-1084-03
Construction and identification of lentivirus of RNA interference
targeting VEGF165 gene
LIU Bei,MA Ge-jia,SONG Bao-qiang
(Department of Plastic Surgery,Xijing Hospital,The Fourth Military Medical University,Xi'an,710032 Shaanxi,China)
Abstract:ObjectiveTo construct a lentiviral vector of RNA interfence (siRNA) of VEGF165 gene, and preparing mature virus.MethodsThe effective sequence of siRNA targeting VEGF165 was confirmed in our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was synthesized and cloned into the PGC SIL-GFP lentivector obtaining the VEGF165 siRNA virus vector named pGCSIL-SiVEGF. 293T cells were cotransfected with pGCSIL-SiVEGF and lentivirus packaging plasmids mixtures to prepare the mature virus named LV-SiVEGF. The titer of virus was tested according to the expression level of GFP.The human umbilical vein endothelial cell (HUVEC) was transfected by the LV-SiVEGF. We detected expression levels of VEGF mRNA of HUVEC by RealTime-PCR.ResultsPCR and DNA sequencing demonstrated that the lentivirus RNAi vector of VEGF was constructed successfully. The titer of concentrated virus was 2×109 ifu/μl. VEGFmRNA expression levels of the experimental HUVEC were significantly lower than the control group (73.3%).ConclusionsVEGFsiRNA lentiviral was successfully constructed.
Key words:VEGF;lentivirus;RNA interference
增生性瘢痕常发生于烧、创伤及手术后，在中国人中有很高的发病率，但目前缺乏理想的治疗措施。而增生期瘢痕具有丰富的血运，许多研究表明血管生成是瘢痕形成的一个重要因素[1-4]。VEGF是最重要的促血管生成因子，我们拟通过抑制血管生成作用抑制瘢痕增生，本实验室在2008年已成功筛选了针对VEGF基因的SiRNA干扰序列，并构建了干扰质粒[5]，但由于质粒的转染效率不高，且不能稳定表达，因此本研究拟构建靶向干扰人VEGF的慢病毒载体。
1材料和方法
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