Whole human source anti-vascular endothelial cell growth factor receptor 2 single chain antibody

A growth factor receptor, fully human antibody technology, applied in the field of bioengineering, can solve problems such as thrombosis

Inactive Publication Date: 2010-10-20
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, blocking VEGF to treat tumors also inhibits the normal physiological function of

Method used

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  • Whole human source anti-vascular endothelial cell growth factor receptor 2 single chain antibody
  • Whole human source anti-vascular endothelial cell growth factor receptor 2 single chain antibody
  • Whole human source anti-vascular endothelial cell growth factor receptor 2 single chain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Screening of fully human anti-human VEGF receptor 2 single chain antibody

[0022] Coating buffer (50mM NaHCO 3 , pH9.6) to dilute KDR3, take 4ml and add it to the immunotube, and coat overnight at room temperature; the next day, discard the supernatant, and wash the tube 3 times quickly with PBS; 2% MPBS (PBS containing 2% skimmed milk), 37°C Block for 2 hours; discard the blocking solution, and wash the tube 3 times quickly with PBS; the phage antibody library (10 12 ~10 13 p.f.u) was suspended in 4ml 2% MPBS and added to the immunotube. After repeated inversion at room temperature for 30 minutes, it was left to stand at room temperature for more than 90 minutes; the tube was washed 10 times with PBS containing 0.1% Tween-20, and then washed 10 times with PBS to remove Remove detergent; add 1ml 100mM triethylamine [700μl triethylamine (7.18mol / L) is added to 50ml water], and incubate repeatedly at room temperature for 10min for specific elution; 0.5ml 1M ...

Embodiment 3

[0023] Example 3. Soluble expression and isolation and purification of anti-KDR single-chain antibody

[0024] Select the correctly sequenced TG1 strain, collect the phagemid, infect the expression strain Escherichia coli HB2151 according to the routine operation, and cultivate to OD 600nm =0.6, add IPTG with a final concentration of 1 mM, induce overnight at 25°C, and detect the result of induced expression by 12% SDS-PAGE.

[0025] 6000rpm, centrifuge at 4°C for 5min, collect the cells; resuspend the cells in PBS, sonicate (sonication 3s, interval 3s, 10min in total); 10000rpm, centrifuge at 4°C for 20min, keep the supernatant; ), eluted with different concentrations of imidazole; 12% SDS-PAGE detected the collected samples, and stored the target protein at -20°C.

Embodiment 4

[0026] Example 4. Western Blot identification of anti-KDR single chain antibody

[0027]The purified AK404R was subjected to denaturing SDS-PAGE electrophoresis, and the concentration of the separating gel was 12%; 4°C, 100mA constant current transfer for 2 hours, and the protein was transferred to a PVDF membrane (purchased from Millipore); after the transfer, the membrane was placed in 5% MTBS (TBS containing 5% skimmed milk) for blocking at room temperature for 1 h; dilute anti-6×His antibody (Novagen product) with 5% MTBS at 1:1000, incubate at room temperature for 1 h, wash 3 times with TBS, 10 min each time; The HRP-anti-Mouse secondary antibody (purchased from Lianke Biotech) was diluted 1:10000 in MTBS, incubated at room temperature for 1 h, washed 3 times with TBS, 10 min each time, and developed with DAB.

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PUM

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Abstract

The invention belongs to the technical field of genetic engineering antibodies and particularly relates to a whole human source single chain antibody which is designed and expressed by genetic engineering and has specific affinity towards human vascular endothelial growth factor receptor 2 (VEGFR-2). The invention also provides a nucleotide sequence of heavy chain variable region and light chain variable region immunoglobulin molecules, a nucleotide sequence which comprises the heavy chain variable region and light chain variable region immunoglobulin molecules, an amino acid sequence which comprises variable heavy chain and variable light chain immunoglobulin molecules, and a sequence which corresponds to CDR1, CDR2 and CDR3 of a complementary determining region. The invention also provides a method for generating and expressing the anti-VEGFR-2 single chain antibody, by cycling operations of gathering-elution-gathering, an antibody which has specific affinity towards the human vascular endothelial growth factor is screened, and then the single chain antibody is obtained through a prokaryotic cell secretion and expression system and an affinity purification system. The antibody can be coupled with a detectable substance and therapeutic agent.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a single-chain antibody capable of specifically binding to human vascular endothelial cell growth factor receptor 2, capable of blocking the binding of VEGF to its receptor, and neutralizing the The biological effects produced by the combination of 2. Background technique [0002] Angiogenesis is the development of new blood vessels, including the proliferation, migration and tissue invasion of vascular endothelial cells, the formation of vascular skeleton by cells, the formation of new blood vessels in the existing circulatory system, and the maturation of new microvessels. Angiogenesis is involved in normal physiological processes including stem cell development, follicle growth and wound healing, pathological conditions such as tumor growth, and abnormal angiogenesis such as glaucoma. Other disease states include, but are not limited to, atherosclerosis, rheumatoid ar...

Claims

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Application Information

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IPC IPC(8): C07K16/28G01N33/577
Inventor 王旻张娟李海鑫陈卫周雅琼
Owner CHINA PHARM UNIV
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