| Thrombosis is one of diseases resulted in death in mankind presently. To prevent this disease from thrombus formation is the key point. Urokinase and streptokinase are currently applied to clinical therapy. However they are prone to hemorrhage because of the limited solving efficiency and the unspecified properties of solving thrombus. Associated with systemic activation of plasminogen they can produce indiscriminate digestion of coagulation proteins, and significantly increase the risk of haemorrhage during treatment. Comparing with UK and SK, t-PA is one kind of natural fibrinolysis substances and has high specific affinity to the thrombus .The inactive proenzyme plasminogen is enzymatically converted into plasmin which is capable of dissolving the fibrin in blood clots and renewing the circulation by tissue plasminogen activators. Since t-PA is not correleated with systemic activation of plasminogen ,it significantly decrease the risk of haemorrhage during treatment. Therefore, it is a drug with specially good effect used to the treatment of acute thrombotic disorders such as myocardial infarction. In addition to revolving in regulating the balance of hemolysis and coagulantion, it is closely concerned to some important physiological and pathological course, such as tissue reforming, cell translocation , ovulation, activated peptide production , tumor invasion and so on .T-PA is widely distributed in the normal tissue, yet the serum level is very low with a content of 4-20μg/L and the half life is very short for only 4-5 minutes. Therefore it is very difficult to obtain t-PA from natural materials for therapy or even for research. It will be a promising way of gaining plenty of t-PA through genetic engineering . Each method for producing recombinant proteins has its own virtues anddefects. The proteins translated in the prokaryote ,such as Escherichia coli ,can not be modified by phosphorylation and acetaminolation because of lacking the modifying enzymes. Thus the proteins can not be secreted because they are folded incorrectly and formed into inactivated occlusion body. Purification of t-PA expressed in yeast is complicated and costly. The production of t-PA can not meet the demands. The retrovial vector can transfere the exogenous gene into cells .Yet, it will activate oncogene and cause virus infection. Construction of eurokaryotic expressing vector is an efficient way of producing exogenous proteins. Both SV40 promoter and CMV promoter of pcDNA3 are prowerful promoters. Our experiment established the eurokaryotic expressing vector by laying t-PAcDNA under the control of SV40 and CMV promoters, eukaryotic expressing vector (pcDNA3-t-PA) was transfected into MCF-7 cells mediated by lipofectamin?000 with Geneticin (G418) selection. This experiment established the basis of producing t-PA in transgenic animalsMarerials and Methods: The t-PAcDNA was amplified by PCR techniques. Theplasmid PETPFR containing t-PAcDNA was used as the templete of PCR . The up and down primers possess HindIII and Sal I site respectively . The PCR fragment of t-PA was digested with both HindIII and Sal I. The agarose gel electrophoresis was used to recover the 2.1kb band of t-PA. pcDNA3 was digested with HindIII and Xho I and 5.4kb fragment was recovered by agarose gel electrophoresis. The sticky ends of Sal I and Xho I were compatible . The 2.1kb fragment of t-PA. and 5.4 kb fragment of pcDNA3 were linked by T4 DNA ligase in vitro at 16℃ overnight. The recombinant was transformed into the competent bacterials JM109 . The transformed bacterials were spreaded onto the culture dish with Amp and cultured in incubator at 37℃ overnight. Ten clones were selected randomly and inoculated in LB culture medium with Amp respectively and cultured at 37℃. The recombinant plasmid was extracted and identifed by enzymes digestion. The 7.5kb fragment will be obtained with HindIII digestion and 2.1kb and 5.4kb fragments with HindIII and Xba I . In this way the positive clones were identified primarily. The insert was finally d...
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