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Functional mutant of human plasminogen, its preparation method and application

A technology of human plasminogen and plasminogen, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the PLM mutation that has not yet had both thrombolytic, anticoagulant and antithrombotic effects Body and other problems, to achieve the effects of refolding, reducing pro-inflammatory side effects, and increasing the possibility

Inactive Publication Date: 2011-09-28
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although a variety of PLM mutants containing SP domains have been constructed and have shown good thrombolytic activity, there are no reports of PLM mutants with both thrombolytic, anticoagulant and antithrombotic effects

Method used

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  • Functional mutant of human plasminogen, its preparation method and application
  • Functional mutant of human plasminogen, its preparation method and application
  • Functional mutant of human plasminogen, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Preparation of hPLG-?K

[0047] 1. Subcloning of the hPLG-?K gene: use the pDNR-LIB-hPLG plasmid containing the full-length cDNA sequence of human PLG as a template, and carry out the first round of PCR with the upstream primer SEQ ID NO:7 and the downstream primer SEQ ID NO:8 , wherein the coding sequence tag of 6 histidine residues is introduced in the upstream primer SEQ ID NO:7, and the downstream primer SEQ ID NO:8 adds Xba The enzyme cutting site of I, reaction conditions: 94°C for 5 min; 94°C for 30 s, 40°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 7 min, PCR product 1.0% agarose gel electrophoresis, Gel recovery, concentration determination. Using the product as a template, carry out the second round of PCR with the upstream primer SEQ ID NO:9 and the downstream primer SEQ ID NO:8, wherein the primer SEQ ID NO:9 contains xho I Restriction site and Kex 2 Enzyme recognition site coding sequence, PCR reaction conditions: 94°C for 5 min; 94°C fo...

Embodiment 2

[0059] Example 2 Preparation of RGD-hPLG-?K

[0060] 1. The construction of the pGME-T-RGD-hPLG-?K plasmid: through three rounds of PCR reactions, the first round uses the pGME-T-hPLG-?K plasmid constructed in Example 1 as a template, and uses the upstream primer SEQ ID NO:9 and downstream mutation primer SEQ ID NO:10 carry out PCR; Eco The RI linearized pGME-T-hPLG-?K plasmid was used as a template, and the first round of PCR fragment was used as an upstream primer, and the downstream primer SEQ ID NO: 8 was used for PCR; the second round of PCR product was used as a template for the third round, and the above Swimming primer SEQ ID NO:9 and downstream primer SEQ ID NO:8. After the PCR products were subjected to 1.0% agarose electrophoresis, gel recovery, and concentration determination, they were connected to the pGME-T vector, transformed into competent Escherichia coli TOP10, screened white spots on Amp-LB culture plates, colony PCR and sequencing identified pGME-T-RGD-h...

Embodiment 3

[0071] Example 3 Preparation of RHP-hPLG-?K

[0072] 1. The construction of the pGME-T-RHP-hPLG-?K plasmid: through three rounds of PCR reactions, the first round uses the pGME-T-hPLG-?K plasmid constructed in Example 1 as a template, and uses the upstream primer SEQ ID NO:9 and downstream mutation primer SEQ ID NO:11 carry out PCR; Eco The RI linearized pGME-T-hPLG-?K plasmid was used as a template, and the first round of PCR fragment was used as an upstream primer, and the downstream primer SEQ ID NO: 8 was used for PCR; the second round of PCR product was used as a template for the third round, and the above Swimming primer SEQ ID NO:9 and downstream primer SEQ ID NO:8. After 1.0% agarose electrophoresis, gel recovery, and concentration determination, the PCR product was connected to the pGME-T vector, transformed into competent E. xho I and Xba I restriction sequencing confirmed that the pGME-T-RHP-hPLG-?K plasmid was constructed successfully.

[0073] 2. Construc...

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Abstract

A functional mutant of human plasminogen disclosed in the invention, respectively, is hPLG-delta K: human plasminogen protein Pro<544>-Asn<791> polypeptide; Pro<559> in RGD-hPLG-delta K: hPLG-delta K is mutated to Asp<559>; and Gly<560> in RHP-hPLG-delta K: hPLG-delta K polypeptide is mutated to His<560>. The invention also discloses a preparation method for the functional mutant. The product is obtained by using a plasmid containing a full-length cDNA sequence of human plasminogen as a template to carry out a PCR to construct a plasmid and using Pichia Pastoris expression. The functional mutant has a dual-function of fibrinolysis and inhibiting platelet aggregation or inhibiting fibrin monomer polymerization.

Description

technical field [0001] The invention relates to the field of biotechnology and pharmaceuticals, in particular to a functional mutant of human plasminogen and a preparation method and application thereof. Background technique [0002] Embolism is one of the important causes of death from cardiovascular and cerebrovascular diseases, and thrombolytic therapy is an effective means. The main thrombolytic drugs currently used clinically, such as tissue plasminogen activator (Tissue-type plasminogen activator, t-PA), urokinase (Urokinase-type plasminogen activator, UK), streptokinase (Streptokinase, SK), etc., Although it shows a certain therapeutic effect, it can only exert thrombolytic effect after activating plasminogen (PLG) in the blood to plasmin (PLM), so the efficiency for old thrombus is low, resulting in a treatment time window. Short, high bleeding complications and other shortcomings. And because there is no antithrombotic function, it is easy to occur the situation o...

Claims

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Application Information

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IPC IPC(8): C12N9/68C12N15/57C12N15/10C12N15/81A61K38/48A61P29/00A61P35/00A61P7/02A61P25/28A61P27/02C12R1/84
Inventor 陈武吴茂材吴敬源杨健忠陈振林黄智慧张鑫涌
Owner GUANGDONG PHARMA UNIV
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