MMP-2特异性RNA干扰真核表达载体的构建

The construction of eukaryotic expression vector of RNA interference specific for MMP-2

目的构建基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)特异的RNA干扰质粒载体,为探讨抑制MMP-2表达在急性冠脉综合征治疗中的意义奠定基础。方法根据GenBank数据库提供的MMP-2基因核苷酸序列,选择设计能转录小发卡结构RNA(Small hairpin RNAs,shRNA)的DNA序列,并与psiSTRIKETM质粒载体连接,构建受控于人RNA聚合酶Ⅲ启动子U6的真核表达载体,使用限制性内切酶PstⅠ酶切鉴定是否为阳性克隆。结果成功构建psiSTRIKETM-MMP2载体,拟进一步采用RNAi技术观察其对MMP-2基因表达的抑制情况,从而研究其在急性冠脉综合征治疗中的作用。结论MMP-2 RNAi真核表达载体被成功构建。

Objective To construct of eukaryotic expression vector of RNA interference specific for MMP-2, and explore the significance of MMP-2 inhibition in treatment of acute coronary syndrome.Methods Genome sequences of MMP-2 gene was retrieved from Genbank and cDNA was designed coding expression of shRNA （small hairpin RNAs） for MMP-2 gene. The cDNA was synthesized and inserted into plasmid psiSTRIKE^TM which was eukaryotic expression vector controlled by the U6 promoter of RNA polymerase Ⅲ, and identified by the restriction map. Results The recombinant plasmid of RNA interference specific for MMP-2 identificated by the restriction map completely coincided with the designs. Conclusion The siRNA eukaryotic expression vector against MMP-2 mRNA has been successfully constructed.