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Trombolysis agent KGD - prourokinase chimaera, preparing process and application thereof

A kind of technology of prourokinase and thrombolytic agent

Inactive Publication Date: 2004-08-18
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The chimeric protein with wild-type prourokinase as a scaffold and chimeric KGD sequence (lysyl-glycyl-aspartyl) has not been reported in domestic and foreign literatures. It is a new type of thrombolytic protein molecule, which is different from existing Homology of other functionally related proteins and peptides

Method used

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  • Trombolysis agent KGD - prourokinase chimaera, preparing process and application thereof
  • Trombolysis agent KGD - prourokinase chimaera, preparing process and application thereof
  • Trombolysis agent KGD - prourokinase chimaera, preparing process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. KGDW-prourokinase gene of chimeric KGDW sequence

[0039] Construction and transfection of insect cell Sf9

[0040] Experimental Materials:

[0041] Strains: E.coli DH5α, BL21(DE3), JM109 are all commercially available products, and PUC19-proUK cDNA was constructed in our laboratory. Insect baculovirus expression system Sf9( Spodoptera frugiperda-9 ) cells and Sf21 cells were purchased from Invitrogen, USA.

[0042] Primers: DNA fragments of oligonucleotide primers were synthesized by Beijing Saibaisheng Company. Primer 1: 5'CCCAAG CTT GAT ATC ATG A3'; Primer 2: 5'GACAAGCGGCTTTA GCCAGTCCCCTTTGCCC ACCTGC ACATA 3'; Primer 3: 5'CTA AAG CCG CTT GTC 3'; Primer 4: 5'GTG GCG CTG ATC ACCC 3' .

[0043]Chemical reagents and enzymes: commonly used restriction enzymes, T4 DNA ligase, Klenow fragment, T4 DNA polymerase, T4 polynucleotide kinase, Taq DNA polymerase, RNase, ATP, dNTPs, etc. were purchased from Promega Company and New England Biolab Company , Hua...

Embodiment 2

[0055] Example 2. Insect cell Sf9 transfected with KGDW-prourokinase chimeric gene

[0056] Culture and induced expression of KGDW-prourokinase chimeric protein

[0057] The KGD-prourokinase chimeric protein was produced by insect cell secreted expression. Firstly, adapt the insect cell Sf9 series under serum-free conditions to obtain insect cells that can grow well in Sf900II serum-free and protein-free medium, and use Sf900II SFM medium for insect cell culture in order to express the target gene Product purification. A sequence before the coding frame of the recombinant KGD-prourokinase gene is responsible for encoding a signal peptide, which assists the expression product of the KGD-prourokinase gene to penetrate the membrane and be secreted into the supernatant of the cell culture medium.

[0058] There are two ways to culture insect cells: adherent culture and suspension culture. Both culture methods can express exogenous genes well. The culture of insect cells re...

Embodiment 3

[0060] Example 3. Separation and purification of KGDW-prourokinase chimeric recombinant protein

[0061] From the perspective of the primary structure, the KGD-prourokinase recombinant protein is basically the same as the wild-type prourokinase, only four amino acid residues are added in the K region of the prourokinase; According to the homology modeling results of the protein structure, KGD-prourokinase basically maintained the three-dimensional structure of the natural prourokinase. The results of Western blotting analysis and antibody neutralization inhibition experiments showed that the KGD-prourokinase recombinant chimera molecule retained the immunodeterminant structure on the prourokinase molecule, and could be recognized and combined by anti-prourokinase antibody. Therefore, using this characteristic, an affinity chromatography column coupled with prourokinase antibody was prepared to purify the target gene expression product KGDW-prourokinase in insect cells by aff...

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Abstract

The present invention relates to new-type thrombolytic containing chimera of KGD sequence and prourokinase and its preparation process and application. Through gene engineering process, short peptide with KGD conservation sequence is embedded into wild prourokinase gene. The preparation process of the thrombolytic includes the steps of; constituting KGD-prourokinase gene with chimeric KGD sequence and transfecting insect cell; culturing the insect cell and expressing the KGD-prourokinase gene in the insect cell; separating purification and identification of the chimeric KGD-prourokinase gene expressing product; and measuring the bioactivity of recombinant protein of chimeric KGD-prourokinase. The chimera of the present invention is one kind multifunctional thrombolytic with thrombolytic effect and thromobocyte fibrinogen receptor specificity and has wide clinical application foreground.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, the preparation of novel thrombolytic agent KGD-prourokinase chimera by genetic engineering technology, its preparation method and application. Background technique [0002] Cardiovascular and cerebrovascular diseases have become a class of diseases that seriously endanger human health in modern society. Including hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis and so on. Abnormal formation of thrombus has always been one of the important causes of disease. In the process of treating such diseases, human beings have been working hard in both prevention and treatment, and have pursued various antithrombotic and thrombolytic agents that are more efficient, specific, and easy to mass-produce as the best way to prevent and treat such diseases. way. The abnormal formation of thrombus in the blood circulation system is often the direct cause of such di...

Claims

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Application Information

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IPC IPC(8): A61K38/49A61P7/02A61P9/10C12N15/62C12N15/86
Inventor 俞炜源井健
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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