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Antibody molecule ATF-Fc fusion albumen of antiurokinase type fibrinolysin activator receptor and use thereof

A technology of fusion proteins and uses, applied in the direction of organic active ingredients, peptide/protein ingredients, medical preparations containing active ingredients, etc., to achieve the effect of long half-life and high affinity

Inactive Publication Date: 2007-02-28
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the development of ATF-Fc fusion protein at home and abroad

Method used

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  • Antibody molecule ATF-Fc fusion albumen of antiurokinase type fibrinolysin activator receptor and use thereof
  • Antibody molecule ATF-Fc fusion albumen of antiurokinase type fibrinolysin activator receptor and use thereof
  • Antibody molecule ATF-Fc fusion albumen of antiurokinase type fibrinolysin activator receptor and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Cloning of embodiment 1.ATF gene and construction of pcDNA3.1 / ATF-Fc eukaryotic expression vector

[0052] Take the pIRES-dhfr / HMW-ProUK vector as a template (this expression vector is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, the preservation date is February 24, 2006, and the preservation number is CGMCC No.1623, the microorganism of reference The strain is pIRES-dhfr / HMW-ProUK, the proposed classification is named Escherichia coli, and the Chinese invention patent application number is 200610065813.4), using primers ATFs and ATFx as upstream and downstream primers, ATF gene is amplified by PCR, and ATF is recovered Gene.

[0053] ATFs: 5'-TGCGCTAGCCCACCATGAGAGCCCTGCTGGCGCG-3'

[0054] ATFx: 5'-CGCGGATCCACTTACCTGTACTGCCAGATCCGCTTCCATCTGCGCAGTCATGCAC-3'

[0055] The PCR reaction system was: 8 μL dNTP (2.5 mmol / L), 0.5 μL Pyrobest polymerase, 2 μL pIRES-dhfr / HMW-ProUK vector, 10 μL 10×Buffer, 2 μL A...

Embodiment 2

[0057] Example 2. Lipofectamine transfection and cell culture

[0058] Lipofectamine produced by Invitrogen was used for transfection TM2000 cationic liposome transfection kit, operate according to the kit instructions. Transfected CHO-K1 cells were cultured in a 5% CO2, 37°C incubator. After 12 hours of transfection, the DMEM / F12 medium (Hyclone) containing 10% enhanced calf serum (Hyclone) was replaced. After 48 hours, use 750 μg / mL G418 (Sigma) selection medium to pressurize and screen, change the medium every 2-3 days, and after pressurizing for about 12 days, digest the resistant cell clones with 0.25% trypsin, and use the limiting dilution method in Monoclonal culture was carried out in a 96-well microwell cell culture plate (NUNC Company), that is, the cells were diluted to 10 cells / mL with DMEM / F12 medium containing 10% calf serum and 300 μg / mL G418, and each microwell The wells were inoculated with 200 μL of culture medium (each well contained about 2 cells on aver...

Embodiment 3

[0060] Embodiment 3. Purification and SDS-PAGE analysis of expressed protein

[0061] The collected ATF-Fc-H6 serum-free culture supernatant was separated and purified by protein A affinity chromatography column (Pharmacia). Using the specific adsorption of protein A to IgG, the expressed protein was purified by affinity chromatography using nProtein A Sepharose 4 Fast Flow separation medium (Amersham Biosciences). See the product description for the operation method. The bound protein was eluted with pH 2.5, 0.1mol / L glycine-HCl buffer, and there was an obvious elution peak (Figure 4(A)), and the eluted components were eluted with 2mol / L Tris-HCl buffer. The pH of the solution was adjusted to 7. After purification, A was determined by UV spectrophotometer 260 and A 280 Calculate the protein concentration by absorbance, and the formula for protein quantification is: protein content (mg / mL) = 1.45×A 260 -0.74×A 280 . The purity and molecular weight of the protein were de...

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Abstract

The invention discloses an antibody molecule ATF-Fc merge protein and application of antiurokinase type profibrinolytic activator acceptor in biological medicine area. which is characterized by the following: the ATF-Fc merge protein is ATF area of uPA and Fc of human immune globulin IgG1; heavy chain constants region 2 and heavy chain constants region 3 through Ser-Gly-Ser-Gly-Ser joint form to dimer merge protein; possessing 742 amino acids; single-chain possesses amino acids sequence of sequence table SEQ ID No.1, wherein the front 20 amino acids is uPA signal peptide.

Description

technical field [0001] The invention relates to an anti-urokinase-type plasmin activator receptor (uPAR) antibody-like molecule ATF-Fc fusion protein and its use in tumor treatment, belonging to the field of biopharmaceuticals. Background technique [0002] Urokinase-type plasminogen activator (uPA) is a protein composed of 411 amino acid residues, including single-chain uPA (scu-PA) without enzymatic activity and double-chain uPA with enzymatic activity uPA (tcu-PA), single-chain uPA, also known as prourokinase (Pro-UK), is activated after the peptide bond between Lys158-Ile159 is broken under the action of plasmin, trypsin and kallikrein Plasminogen-functional double-chain uPA (ie urokinase, UK). [0003] Natural uPA is a glycoprotein with a molecular weight of about 52-54kD composed of 411 amino acids, with 12 pairs of disulfide bonds, an N-glycosylation site (Asn302) and an O-glycosylation site (Thr18), It has three protein domains (Figure 1): (1) epidermal growth fact...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N1/21A61K38/17A61P35/00A61K31/7088
Inventor 胡显文高丽华段海峰陈惠鹏陈金龙殷亮潘淑媛郗永又
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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