抗PAI抑制作用的组织纤溶酶原激活剂在大肠杆菌中的表达

目的:构建t-PA活性不被PAI-1抑制的新一代t-PA突变体。方法:根据t-PA的结构特点,去除t-PA分子中的指状区、表皮生长因子和Kringle1区,以含全长t-PA编码区序列的pUC18质粒为模板,经PCR扩增编码氨基酸1~3和176~527位的截短式t-PADNA序列;并将该t-PA分子中的PAI-1结合位点,即第373~384位核苷酸(AAGCACAGGAGG)突变为(GCGGCCGCGGCG),相应的氨基酸KHRR则变为AAAA。结果:测序证实,t-PA突变体的DNA序列正确,将其克隆于大肠杆菌表达载体中,并在大肠杆菌中得到高效表达。表达蛋白占总菌体蛋白的30%,以包涵体形式存在。经蛋白质变性、复性,得到有活性的t-PA突变体。t-PA突变体与PAI-1反应后t-PA的活性未受到抑制。结论:t-PA突变体可能是一种用于治疗心肌梗死和脑血栓等血栓性疾病的强效且剂量要求低的新型生物工程药物。

Expression and activity of tissue-type plasminogen activator mutant reteplase with deletion of PAI-1 binding sites

AIM: To construct a mutant of human tissue plasminogen activator (t-PA) with activity not inhibited by plasminogen activator inhibitor-1 (PAI-1). METHODS: The finger, epidermal growth factor and kringle-1 domains were removed from native t-PA to obtain a t-PA mutant with the PAI-1 binding sites. A pUC18 plasmid containing the human t-PA full-length cDNA sequence was used as template, and the DNA sequences encoding 1-3 and 176-527 amino acids were amplified by PCR. The nucleotides AAG CAC AGG AGG (from 373 to 384) in the PAI-1 binding site changed to GCG GCC GCG GCG, so amino acid KHRR was replaced by AAAA correspondently. RESULTS: Sequencing result showed that the above t-PA mutant DNA sequence was consistent with the expected. Then it was cloned in an E.coli expression vector and was highly expressed. The expressed protein occupied about 30% of total bacterial proteins as inclusion body. After denaturation and renaturation, the active t-PA mutant was obtained, whose activity was not inhibited by PAI-1. CONCLUSION: This t-PA mutant may be developed to a new gene-engineering drug with higher therapeutic activity and lower dose requirement in the treatment of acute myocardial infarction and cerebrovascular thrombosis diseases.