HeLa细胞中编码人组织型纤溶酶原激活物K2P结构域突变体cDNA的克隆及特性

Cloning and Characterization of cDNA Coding for a Mutant of Human Tissue-Type Plasminogen Activator K2P Domain from HeLa Cells

组织型纤溶酶原激活物(t-PA)在人体纤溶系统中发挥着重要作用。基因重组t-PA已被用于急性心肌梗死等血栓性疾病的溶栓治疗。另外,t-PA还与肿瘤浸润和转移等多种过程有关,因此对t-PA进行功能结构研究具有重要应用价值和理论意义。本实验用RT-PCR方法从豆蔻酰佛波醇乙酯(phorbol myristate acetate,PMA)处理的HeLa细胞扩增并克隆获得了人t-PA K2P结构域的编码cDNA。序列分析显示该序列含有两个碱基突变,分别导致了成熟蛋白分子两个氨基酸践基序列的改变。第263位密码子突变(ACC→GCC)导致Thr^(263)→Ala^(263),另一突变(CTG→CCG)导致Leu^(485)→Pro^(485)。两个突变分别位于K2和P结构域的连接区和P区。将此含有突变的K2Pt-PA cDNA重组到原核表达载体,在大肠杆菌中获得了表达。纤维蛋白平板法检测显示重组K2Pt-PA复性后具有纤溶活性。以上结果表明HeLa S3细胞表达一种具有纤溶活性的t-PA突变体。

The serine protease tissue-type plasminogen activator (t-PA) plays an important role in the process of fibrinolysis as well as in cancer invasion and metastasis. To study the structure and function of t-PA, cDNA coding K2P domain of human t-PA was cloned from HeLa S3 cells treated with phorbol myristate acetate (PMA) using RT-PCR method. Sequence analysis revealed variant nucleotides compared with those reported for the cDNAs and genomic DNAs derived from other human cells. There are two single base substitutions occurring in the coding sequence, one of which showed an GCC triplet coding for Ala263 instead of ACC coding for Thr 263, another showed CCG coding for Pro485 instead of CTG coding for Leu485. Production of this t-PA mutant in Escherichia coli led to the formation of inclusion bodies, from which fibrinolytically active recombinant protein was generated by an in vitro refolding process. These results showed that HeLa S3 cells express a kind of t-PA mutant with fibrinolytic activity.