Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Low molecular-weight urokinase mutant and its expression vector

A low-molecular-weight, urokinase technology is applied in the direction of introducing foreign genetic material, enzymes, and enzymes by using carriers, which can solve problems such as lack of enzyme catalytic activity, improve specific activity, eliminate thrombin enzyme cleavage sites, and reduce drug dosage. Effect

Inactive Publication Date: 2008-12-10
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
View PDF7 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the Arg 156-Phe157 peptide bond of pro-UK can be hydrolyzed by thrombin to produce a double-chain molecule connected by disulfide bonds, and its activity is only 1 / 500 of that of double-chain UK. The original has almost no enzymatic activity

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Low molecular-weight urokinase mutant and its expression vector
  • Low molecular-weight urokinase mutant and its expression vector
  • Low molecular-weight urokinase mutant and its expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The construction of embodiment 1.pIRES-dhfr eukaryotic expression vector

[0040] Using the pUC19 / dhfr plasmid as a template, primers dhfr1 and primer dhfr2 as upstream and downstream primers, PCR amplifies the dhfr gene, and the PCR reaction system is: 8 μL dNTP (2.5 mmol / L), 0.5 μL Pyrobest polymerase, 10 μL ATR (1-18) template , 10 μL 10×Buffer, 2 μL dhfr1 upstream primer (10 μmol / L), 2 μL dhfr2 downstream primer (10 μmol / L), 67.5 μL water, the total reaction system is 100 μL, PCR reaction conditions: 94 ° C, 2 min (pre-denaturation) → [ 94°C, 30s→60°C, 60s→72°C, 1min]×25 cycles→72°C, 5min→4°C, 5min. The PCR product was recovered with 0.8%-1% low melting point agarose gel (Promega, Agarose LMP).

[0041] The dhfr gene recovered by the PCR product recovery kit was double-digested with Xma I and Not I, and the pIRES vector was double-digested with XmaI and Not I, and the above two pieces of DNA were ligated into a pIRES-dhfr expression vector under the action of T4 li...

Embodiment 2

[0044] Example 2. Construction of low molecular weight urokinase eukaryotic expression vector

[0045] 1. Method:

[0046] Using site-directed mutagenesis, Arg156 in the natural high molecular weight prourokinase molecule was mutated to Lys to eliminate the thrombin cleavage site, and Asn302 was mutated to Ala to remove the glycosylation site, and the HMW-proUK mutant The gene was inserted into the pIRES-dhfr vector to obtain the vector pIRES-dhfr / HMW-proUK (CGMCC No.1623) expressing the thrombin-resistant non-glycosylated HMW-proUK mutant.

[0047]In order to make LMW-UK secreted and expressed, the signal peptide of pro-UK was introduced upstream of its coding gene, the specific method is: (1) PCR overlap extension primer HMW1 and primer SIG generate pro-UK signal peptide sequence LMWfrag1, overlap extension PCR reaction The system is: 2 μL of dNTP (2.5 mmol / L), 0.125 μL of Pyrobest polymerase, 2.5 μL of 10×Buffer, 5 μL of oligonucleotide fragments HMW1 and SIG (5 μmol / L), 1...

Embodiment 3

[0057] Example 3. Lipofectamine transfection and cell culture

[0058] 1. Method:

[0059] Lipofectamine TM 2000 cationic liposome transfection kit, operate according to the kit instructions. Transfected CHO-dhfr - Cell culture in 5% CO 2 , 37 ℃ thermostat. After 12 hours of transfection, the DMEM / F12 medium containing 10% enhanced calf serum was changed. After 48 hours, use DMEM screening medium containing 3% calf serum and 200nmol / L MTX (Sigma) to screen under pressure, change the medium every 2d, and increase the concentration of MTX to 1000nmol / L after pressurizing for about 10d, continue Pressurized culture for 7-10d. Afterwards, continue culturing with DMEM / F12 medium (Hyclone) containing 5% enhanced calf serum, digest with 0.25% trypsin after growing out of MTX-resistant cell clones, and use the limiting dilution method in 96-well microwells Monoclonal culture was carried out in a cell culture plate (NUNC Company), and cell clones appeared after 2 weeks. Take th...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
concentrationaaaaaaaaaa
Login to View More

Abstract

The invention discloses a low-molecular weight urokinase mutant and expressing carrier in the gene engineering domain, which is characterized by the following: possessing amino acid sequence as sequence list SEQ ID No.1 displayed; improving product stability and uniformity to control quality; lengthening drug half-decay time in vivo; improving specific activity of protein; fitting for biological technological drug manufacturing for mammal cell.

Description

technical field [0001] The invention relates to a low molecular weight urokinase mutant and its expression carrier, belonging to the field of biopharmaceuticals. Background technique [0002] Thrombosis such as myocardial infarction, cerebral thrombosis and pulmonary embolism is a common disease that seriously endangers human health. In developed countries, cardiovascular and cerebrovascular diseases have become the diseases with the largest number of deaths. The number of deaths from cardiovascular and cerebrovascular diseases in my country is second only to Cancer, ranked second. Urokinase (UK) is the most commonly used thrombolytic drug in my country, but currently there is no recombinant human urokinase product approved for marketing in the world. The urokinase products approved for marketing in my country are mainly extracted from human urine, and the US FDA approved it for marketing in 2002 Abbott (Abbott) low molecular weight urokinase It is a product of human fetal ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/72C12N15/58C12N15/85C12N5/10
Inventor 胡显文高丽华陈惠鹏潘淑媛殷亮杨波张正光
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com