hWAPL基因shRNA真核表达载体的构建及干扰效果的初步鉴定  

Transfection of eukaryotic expression vector expressing shRNA targeting human WAPL in vitro

作　　者：曹利仙[1] 潘巍巍[1] 黄玉蓉[1] 张欣怡[1] 康燕琴[1] 

机构地区：[1]嘉兴学院医学院解剖学教研室,嘉兴314001

出　　处：《解剖学杂志》2010年第4期446-450,共5页Chinese Journal of Anatomy

基　　金：浙江省实验动物科研计划项目（2008F80025）;嘉兴市科技局科研项目（2008AY2039-3）

摘　　要：目的：构建针对人半翼基因（hWAPL）的短发夹RNA（shRNA）真核表达载体,并观察其转染效率.方法：根据hWAPL基因序列,设计并合成特异性的小干扰片段,将其定向克隆到带有卡那抗性和增强绿色荧光蛋白的真核表达载体pGenesil-1中,酶切和测序后,用脂质体转染到CasKi细胞中,观察荧光表达,鉴定转染效率.RT-PCR检测结果表明,筛选到的短链hWAPL siRNA能有效抑制内源hWAPL的表达.结果：成功构建了表达shRNA的质粒及其阴性对照质粒,转染到CasKi细胞系中可以特异下调hWAPL mRNA表达,载体转染效率达50%以上.结论：RNAi能特异地下调CasKi细胞中hWAPL mRNA含量,抑制宫颈癌细胞恶性增殖,提示hWAPL可能成为宫颈癌基因治疗的一个新的靶点.Objective： To construct eukaryotic expression vector expressing short hairpin RNA （shRNA） targeting human wing apart-like （WAPL） and to observe their transfection efficiency. Methods： According to the sequence of human WAPL gene, the otigonucleotides of shRNA were designed and synthesized and cloned into plasmid pGenesil-1. The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing. The recombinant vectors were transfected into CasKi cell line by lipofectamine 2000. The expression of fluorescence and efficiency of transfection was detected under a fluo- rescence microscope. The mRNA level of hWAPL was detected by RT-PCR. Results ： Four shRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into CasKi cells and the transfeetion efficiency achieved about 50 %. The vector of hWAPL RNAi specially knocked down the mRNA level of hWAPL gene. Conclusion： The mRNA level of hWAPL in CasKi cell is specially knocked down by RNAi, and the malignant proliferation of cervical cancer cells is inhibited too.

关 键 词：人半翼基因 短发夹 RNA 细胞转染 

分 类 号：R512.62[医药卫生—内科学]