t-PA cDNA基因的克隆及测序

Cloning and sequencing of t-PA cDNA gene

从基因工程菌株里氏木霉中提取基因组DNA,根据t-PAcDNA序列设计一对引物,利用PCR法扩增目的基因,将其与pMD18-T-Vector连接后转入大肠杆菌,通过Amp抗性和蓝白斑筛选出重组菌株,PCR法和双酶切法鉴定后再进行测序鉴定,测序结果表明:克隆的t-PAcDNA的第34位、450位和521位基因发生了突变。

This paper studied the cloning and sequencing of t-PA cDNA. The gene of coding t-PA was amplified by the PCR technique,using Tnchoderma reesei chromosome DNA as a template. The products of PCR were inserted into pMD18-T-Vector, and transfered into Ecoli DH5 α .The recombinant strains were selected by ampicillin resistivity and blue-white reaction.A positive strain cloned t-PA cDNA was identified by restriction enzyme analysis and PER technique.The cloned t-PA cDNA was sequenced.and the result showed that the cloned t-PA cDNA sequence homology was 99.81% with the t-PA eDNA published.