| Thrombosis is one of diseases resulting in death in mankind; and urokinase and streptokinase are currently applied to clinical therapy. However, they have side reaction, such as hemorrhage due to the limited solving efficiency and the unspecific properties of solving thrombus. Comparing with UK and SK, tissue-type plasminogen activator (tPA) is one kind of natural fibrinolysis substances and possesses high specific affinity to the thrombus. The tPA can convert locally the inactive plasminogen into plasmin which is capable of dissolving the fibrin in blood clots and renewing the circulation. Since tPA is not correlated with systemic activation of plasminogen, it significantly decreases the risk of haemorrhage during treatment. Therefore, it is a drug with special effect used to the treatment of acute thrombotic disorders, such as myocardial infarction. In addition to revolving in regulating the balance of hemolysis and coagulation, it is closely concerned to some important physiological and pathological courses, such as tissue reforming, cell translocation, ovulation, activated peptide production, tumor invasion and so on.tPA is widely distributed in the normal tissue, yet the serum level is very low with a content of 4-20ug/L and the half life is very short for only 4-5 minutes. Therefore it is very difficult to obtain tPA from natural materials for research and therapy. It will be a promising way of gaining plenty of tPAthrough genetic engineering. Each method for producing recombinant proteins has its own virtues and defects. 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 recombinant protein expressed in yeast is complicated and costly. The purity of produc can not meet the demands. The retroviral vector can transfer the exogenous gene into cell. Yet, it may activate oncogene and cause virus infection. According to the clinical dosage of tPA (80-1 OOmg/each t ime), p roduction t PA from m edium o f t he c ell 1 ine f or highly effective expression of tPA also can't satisfy the clinical demand.Mammary gland bioreactor technique is to establish the transgenic animals by using the mammalian mammary-specific promoter, which can express the target gene in mammary gland and produce the recombinant protein in milk. Using mammary gland bioreactor to produce recombinant protein possesses virtues of simple expressional system, post-translation modification, low cost and so on. The mammary gland bioreactor has been the first choice for production of recombinant protein.The key point of establishment of mammary gland bioreactor is the construction of mammary-specific vector. The mammary-specific vector is composed of mammary-specific promoter, target gene and poly adenylation signal. The milk protein genes which have been cloned and used to construct vector m ainly ine lude b eta-lactoglobulin gene (BLG), c asein gene a nd whey acidic protein (WAP) gene and etc. In this paper, the cloned 2.4kb WAP promoter was inserted into pBluescript plasmid to construct pW plasmid firstly. Then, the cloned 0.37kb polyadenylation signal was inserted into the downstream of WAP promoter to construct vector pWA. Finally, the cloned target gene 2.1kb tPA cDNA was ligated between WAP promoter and polyadenylation signal to construct mammary-specific vector pWTA. The successfully constructed pWTA expression vector was transfected intomammary carcinoma cell line MCF-7 mediated by Lipofectamine?000 and into mouse mammary glands by direct injection. The expression level of pWTA was identified by transient expression of tPA in MCF-7 cell line and in lactating mice mammary glands demonstrated in situ hybridization.Materials and methods:1. Construction of pW vector: The WAP promoter DNA fragment was amplified by PCR using the Wap2hGH plasmid containing WAP promo...
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