大鼠基质金属蛋白酶抑制因子-1 pRNAT-U6.2小干扰RNA表达载体的构建

Construction of eukaryotic express plasmid of matrix metalloproteinase inhibitor-1 pRNAT-U6.2 siRNA of rat

目的：构建大鼠基质金属蛋白酶抑制因子-1（TIMP-1）小干扰RNA的真核表达质粒pRNAT．U6．2 siRNA。方法：依据siRNA设计原则确定以序列447～465nt、522～540nt、142～160nt为TIMP-1 siRNA靶序列，体外分别合成两端含BamHI和XhoI酶切位点的编码短发夹RNA序列的DNA单链，退火后克隆于pGEM—T载体，r17和SP6为引物进行PCR鉴定；双酶切pGEM—T将其定向克隆到siRNA表达载体pRNAT—U6．2，用pRNAT—U6．2的插入鉴定引物进行PCR鉴定，阳性重组质粒测序。结果：DNA测序证实合成的并被克隆人真核表达载体pR—NAT．U6．2的siRNA插入序列与设计完全符合。结论：TIMP-1 siRNA表达载体构建成功，为后期研究TIMP-1 pRNAT—U6．2 siRNA作用、意义及效果奠定了实验基础。

Aim ： To construct an eukaryotic express plasmid of matrix metalloproteinase inhibitor （ TIMP）-1 pRNAT- U6.2 siRNA of rat. Methods：Lone DNA with cutting sites of BamH I and Xho I enzyme was choiced and combined according the sequence of 447 -465 nt,552 -540 nt, and 142 - 160 nt on TIMP-1 of rat. And pGEM-T vector was cloned,then identified by PCR with T7/SP6 promoter, pGEM-T was cut in sites of BamH I and Xho I enzyme and the fragment to pRNAT-U6. 2 expressing vector was sub-cloned,and identified by PCR and sequencing. Results： RT-PCR and sequencing confirmed that it was succeeded in constructing recombinant plasmid pRNAT-U6.2 siRNA of TIMP-1. Conclusion ： A recombinant plasmid TIMP-1 pRNAT-U6.2 siRNA of rat was constructed successfully and lay the foundation for further study RNAi in TIMP-1.