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

作　　者：徐华锋[1] 邹海峰[1] 刘洋[1] 林平[1] 司云峰 于洪伟[1] 于晓光[1] 

机构地区：[1]哈尔滨医科大学生物化学与分子生物学教研室,黑龙江哈尔滨150086 [2]哈尔滨市红十字中心血站,黑龙江哈尔滨150056

出　　处：《哈尔滨医科大学学报》2005年第2期130-132,共3页Journal of Harbin Medical University

摘　　要：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.

关 键 词：TIMP-1 T7启动子 RNAI DSRNA 

分 类 号：R-332[医药卫生] Q78[生物学—分子生物学]