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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。
OBJECTIVE: To construct a new generation of t-PA mutant whose t-PA activity is not inhibited by PAI-1. Methods: According to the structural characteristics of t-PA, the finger, epidermal growth factor and Kringle1 region of t-PA were removed. The pUC18 plasmid containing the full-length t-PA coding region was used as template to amplify the encoded amino acids 1 to 3 and 176 to 527; and mutating the PAI-1 binding site (AAGCACAGGAGG) of the t-PA molecule to (GCGGCCGCGGCG) , The corresponding amino acid KHRR becomes AAAA. Results: Sequencing confirmed that the DNA sequence of t-PA mutant was correct, cloned into E. coli expression vector and highly expressed in E. coli. The expressed protein accounted for 30% of the total bacterial proteins, in the form of inclusion bodies. By protein denaturation, refolding, active t-PA mutant. The t-PA activity was not inhibited after t-PA mutants reacted with PAI-1. CONCLUSION: The t-PA mutant may be a potent, low-dose, novel bioengineered drug for the treatment of thrombotic diseases such as myocardial infarction and cerebral thrombosis.