甲胎蛋白特异性小干扰RNA表达载体的构建及鉴定

Construction and Identification of Expression Vector of siRNA Specific for Alpha-Fetoprotein

目的:构建特异性抑制甲胎蛋白(AFP)的小干扰RNA(siRNA)表达载体,为进一步研究AFP基因功能及AFP相关肿瘤的基因治疗奠定基础。方法:设计并合成AFP特异性的短链寡核苷酸,退火形成双链DNA片段,通过与RNAi-Ready pSIREN-DNR-DsRed-Express Donor Vector连接、转化大肠杆菌、扩增、纯化得到所需质粒,用琼脂糖凝胶电泳及基因测序鉴定其分子量及插入片段的序列。结果:琼脂糖凝胶电泳证实纯化后的质粒大小约为6 740 bp,测序证实插入序列与合成的寡核苷酸序列完全符合。结论:构建了AFP特异性的siRNA表达质粒pSIREN-DNR-DsRed-Express Donor Vector-AFP。

Objective： To constructα-fetoprotein（AFP） small interference RNA（siRNA） expression plasmid for the research of the function of AFP and the gene therapy of AFP related tumors with RNA interference （RNAi） technology. Methods： AFP specific oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form the double strand DNA fragments and this fragment was cloned into RNAi-Ready pSIREN-DNR-DsRed-Express Donor vector. The plasmid was transformed into Escherichia coli DH5α bacteria to amplify and then purified. The purified plasmid was identified by gel electrophoresis and sequencing. Results：The resuhs of gel electrophoresis and sequencing showed that the RNAi-Ready pSIREN-DNR-DsRed-Express Donor vector were successfully constructed and the sequence was identical with what inserted in, and there was no aberrations such as mutation, deletion, or insertion. Conclusion： Three different expression vectors of siRNA specific for AFP gene has been constructed successfully.