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Prepn, activity detection method ad disease treating application of recombinant human prourokinase amino terminal fragment

A technology for the determination of activity and urokinase, which is applied in the preparation of the amino-terminal fragment of recombinant human pro-urokinase, the determination of activity and its application in disease treatment, which can solve the problems of short survival and poor prognosis

Inactive Publication Date: 2005-12-14
南京大学生物制药工程研究中心
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Problems solved by technology

A large number of studies have shown that the expression of uPAR on the surface of tumor cells is significantly higher than that of normal cells [16], and tumor patients with high expression of uPA and uPAR have poor prognosis and short survival time [17]

Method used

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  • Prepn, activity detection method ad disease treating application of recombinant human prourokinase amino terminal fragment
  • Prepn, activity detection method ad disease treating application of recombinant human prourokinase amino terminal fragment
  • Prepn, activity detection method ad disease treating application of recombinant human prourokinase amino terminal fragment

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Embodiment Construction

[0026] The invention provides a method for preparing the amino terminal fragment of recombinant human prourokinase. Firstly, the total RNA of human umbilical cord vein endothelial cells was extracted, and the amino-terminal fragment gene of human prourokinase was amplified by RT-PCR, and then coupled with the vector after double enzyme digestion. After transformation, identification of positive clones and sequencing, E. coli BL21 (DE3) was used to express the amino terminal fragment of recombinant human prourokinase. Afterwards, its vitality was measured. It is found that the recombinant human prourokinase amino-terminal fragment produced by this method can inhibit the binding of prourokinase and its cell surface receptor, and can inhibit the activation of prourokinase by plasmin and the activation of plasminogen by urokinase.

[0027] Examples of the present invention:

[0028] 1.1 Material

[0029] 1.1.1 Strains and plasmids Human umbilical cord vein endothelial cell line (HUVEC...

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Abstract

The present invention relates to the preparation process and activity identification of one kind of recombinant protein of mutual action of prourokinase (proUK) and prourokinase receptor (uPAR). Owing to that uPA / uPAR takes important role in many pathologic physiology processes, blocking off this passage will inhibit the occurrence and development of diseases. The extracorporeal activity identification shows that the human prourokinase amino terminal fragment (ATF) of the present invention can inhibit the combination between prourokinase and prourokinase receptor on the surface of cell, and inhibit the activation of UK on plasminogen and the activation of plasmin on proUK. This shows that rhATF may take important role in inhibiting the uPA / uPAR participated pathologic physiology processes.

Description

Technical field [0001] The invention relates to the production of a protein fragment capable of inhibiting the interaction between proUK / UK and uPAR by genetic engineering method and its application in disease treatment. Background technique [0002] Cell migration is a periodic process of cell adhesion and de-adhesion. Cell migration occurs in various physiological and pathological processes such as growth and development, tissue formation, differentiation, inflammatory response, wound repair, atherosclerosis, tumor angiogenesis, tumor cell invasion and so on. In recent years, some studies have shown that uPA / uPAR plays an important role in cell migration [1]. uPAR-mediated cell surface protein lysis and non-protein lysis is the center of cell migration and chemotaxis [2]. The combination of uPA and uPAR induces the binding of uPAR and vitreous protein, regulates the interaction between uPAR and integrin or promotes the intracellular signal transduction cascade, promotes the adh...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/72
Inventor 刘建宁陈新园孙自勇朱镇华
Owner 南京大学生物制药工程研究中心