神经元表达发育下调基因siRNA表达载体的构建及鉴定

Construction and identification of a eukaryotic expression vector for small interfering RNA targeting NEDD9 gene

目的:构建针对神经元表达发育下调基因9(Neural precursor cell expressed,developmentally downregulated 9,NEDD9)的小干扰RNA(siRNA)表达载体,研究载体介导的RNAi技术对A549肺腺癌细胞株中NEDD9表达的抑制作用。方法:根据GenBank提供的NEDD9基因(NM_182966)核苷酸序列及siRNA的设计原则,筛选得到159-177 nt、193-211 nt和648-666 nt 3个19 bp片段靶序列,同时随机组合出一条无关序列;分别合成4对带有BamHⅠ、XhoⅠ酶切位点的发夹结构寡核苷酸序列,经退火合成互补DNA双链,克隆入载体pRNAT-CMV3.2中构建重组表达载体,转化DH5α,提取质粒,应用PCR技术和测序方法鉴定重组克隆。用脂质体介导转染入A549肺腺癌细胞株,采用QRT-PCR和Western blot方法检测细胞NEDD9基因mRNA和蛋白表达。结果:PCR筛选得到4个目的片段的阳性克隆,测序证实重组表达载体中插入序列与设计一致。RTPCR和Western blot检测显示构建的siRNA表达载体可显著降低A549细胞中NEDD9 mRNA和蛋白水平的表达。结论:成功构建载体介导的NEDD9靶向RNA干扰重组体,并能有效抑制A549细胞中NEDD9基因的表达。

Objective：To construct a eukaryotic expression vector for the small interfering RNA （siRNA） targeting Neural pre- cursor cell expressed, developmentally downregulated 9 （NEDD9） gene, and to investigate the effects of RNAi on NEDD9 expression in A549 cells. Methods： We used the siRNA design and analyzed software to design the target oligonucleotides according to the sequence of NEDD9 mRNA available in GenBank. Three siRNA sequences were obtained, and the corresponding cDNAs were synthesized and in- serted into plasmid pRNAT-CMV3, l for constructing the recombinant plasmids, which were transformed into E. coli DH5cL strain. The plasmids, after identification by PCR and DNA sequencing, were transfected into A549 cell line via liposome method. The positive cell clones were screened with G418, and the stable transfection and expression of NEDD9 mRNA and protein in the ceils were determined by RT-PCR and Western blot, respectively. The pRNAT-CMV3.2 transfected plasmid was used as control. Results： Four recombinant plasmids were identified by PCR and sequence analysis, the results of which showed correct insertion of the designed sequences in the plasmids. QRT-PCR and Western blot showed substantially decreased mRNA and protein expressions of NEDD9 gene in the transfected cells, as compared with the control group. Conclusion： The recombinant plasmid expressing the siRNA targeting NEDD9 gene has been successfully constructed, siRNA expression vector can inhibit the expression of NEDD9 in A549 cells.