PGEM-T/TIMP-1重组质粒的构建及其在体外转录法合成dsRNA中的作用

Construction of recombinant plasmid PGEM-T/TIMP-1 and the effect on transcription of dsRNA in vitro

目的 构建大鼠PGEM -T/TIMP 1重组质粒,获得TIMP -1基因的dsRNA。方法 RT PCR获得TIMP 1cDNA ,克隆于PGEM- T载体上,PCR鉴定并进行序列分析;以PGEM- T/TIMP- 1重组质粒为模板,PCR获得带有T7启动子的TIMP 1cDNA ;体外转录获得TIMP- 1dsRNA。结果 RT- PCR及测序分析证明成功构建PGEM- T/TIMP 1重组质粒;并获得带有T7启动子的TIMP- 1cDNA以及TIMP- 1dsRNA。结论 成功构建PGEM T/TIMP -1重组质粒,并获得TIMP -1dsRNA ,为RNAi实验奠定了基础。

Objective To construct a recombinant plasmid PGEM-T/TIMP-1 of rat and to obtain the dsRNA of TIMP-1.Methods cDNA of TIMP-1 was obtained by RT-PCR,and coloned PGEM-T vector,and identified by PCR and sequencing;and coloned recombinant plasmid PGEM-T/TIMP-1 was used as template to obtain cDNA of TIMP-1 with T7 promoter by PCR;and dsRNA of TIMP-1 was obtained by transcription.Results RT-PCR and sequencing confirmed that we succeed in constructing recombinant plasmid PGEM-T/TIMP-1;obtaining cDNA of TIMP-1 with T7 promoter and the dsRNA of TIMP-1.Conclusion We construct a recombinant plasmid PGEM-T/TIMP-1 of rat successfully and obtain the dsRNA of TIMP-1,and lay the foundation for further study RNAi.