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Anti to human alkaline fibroblast growth factor human s c F v antibody and application thereof

A technology of fibroblasts and growth factors, applied in the direction of anti-growth factor immunoglobulins, applications, antibodies, etc., can solve the problems of no anti-bFGF human single-chain antibody reports, etc., to achieve inhibition of growth and metastasis, high affinity effect

Active Publication Date: 2012-07-11
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there are no other reports about anti-bFGF human single-chain antibody (ScFv) at home and abroad

Method used

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  • Anti to human alkaline fibroblast growth factor human s c F v antibody and application thereof
  • Anti to human alkaline fibroblast growth factor human s c F v antibody and application thereof
  • Anti to human alkaline fibroblast growth factor human s c F v antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Screening method for anti-recombinant basic fibroblast growth factor (bFGF) or fibroblast growth factor 2 (FGF-2) human scFv antibody:

[0048] (1) Screening of phage antibody library

[0049] Firstly, bFGF (Beijing Qiwei Yicheng Company) was diluted to 50-100 μg / ml with 0.05mol / L, pH8.7 carbonate buffer solution, and 1ml was used to coat the immunotube (Immunotube, NUNC Company), overnight at 4°C On the next day, fill the immunotube with 2.5% skimmed milk powder by mass volume ratio and block at 37°C for 2 hours, pour off the blocking solution, and add 1ml of phage antibody library (about 10 13 CFU) (Enprobiotech Co.), incubate at 37°C for 2 hours, remove the phage antibody solution, and wash twice with PBS (0.01M, pH7.2) containing 0.05% Tween 20 by volume (the second round of washing is 10 times, and subsequent washing more than 20 times), washed once with distilled water.

[0050] Then use two methods to recover the phage antibody bound to the solid phase: ① First...

Embodiment 2

[0058] (1) Prokaryotic expression of single chain antibody

[0059] The positive clones with correct sequencing were amplified by the following primers, and then ligated into the prokaryotic expression vector pET32a (Promega).

[0060] The reaction conditions for PCR are:

[0061] The reaction system is 50 μl:

[0062] Amplify the upstream primer 5'-GGATCCCAGGTGCAGCTGCAGGA-3' of ScFv;

[0063] Downstream primer: 5'-AAGCTTGCTGACCGTCCTAGGG-3'

[0064] 10×PCR Buffer (Mg 2+ ) 5 μl, dNTP Mixture (2mM) 5 μl, upstream and downstream primers 1 μl (10 μmol / L), Blendtaq-plus (Promega Company) 0.5 μl, positive phage antibody clone 1 μl, sterilized deionized water supplemented to 50 μl.

[0065] The PCR amplification program is:

[0066] Pre-denaturation at 94°C for 4min, denaturation at 94°C for 45s, annealing at 54°C for 40s, extension at 72°C for 60s, and after 28 cycles, extension at 72°C for 10min. The reaction product was subjected to 1.2% agarose gel electrophoresis, and the ...

Embodiment 3

[0074] (1) Human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC) proliferation inhibition test

[0075] Use 2×10 3 Inoculate HUVEC cells (Guangzhou Yaji Biotechnology Co., Ltd.) into a 96-well plate at the cell density per well, add 10% fetal bovine serum (FBS, Gibico Company) with M199 medium (Guangzhou Yaji Biotechnology Co., Ltd.) at 37 ° C Incubate for 16 hours. The medium was replaced with M199 medium containing 0.5% FBS, 20 ng / mL bFGF and different concentrations of the human ScFV antibody prepared in Example 1 were added, and the control group was also replaced with M199 containing 0.5% bovine serum (FBS). Medium, add 20ng / mL bFGF and 0.01M, pH7.2 PBS equal to the volume of the antibody), culture at 37°C for 4 days, and use the CCK-8 kit to measure the number of viable cells. see results image 3 , image 3 The results showed that the anti-bFGF ScFv antibody can effectively inhibit the proliferation of HUVEC vascular endothelial cell...

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Abstract

The invention discloses a human single-chain antibody fragment (scFv) antibody of anti-recombination basic fibroblast growth factor and an application thereof. An amino acid sequence of heavy chain variable area of the human scFv antibody is shown in SEQ ID NO. 1, and an amino acid sequence of light chain variable area of the human scFv antibody is shown in SEQ ID NO. 2. A gene sequence encoding the heavy chain variable area is shown in SEQ ID NO. 3. A gene sequence encoding the light chain variable area is shown in SEQ ID NO. 4. The human scFv antibody has the characteristics of high affinity and specificity, and can be directly developed to be used as an antibody drug for human because the human scFv antibody is fully human antibody. The gene encoding the human scFv antibody can be constructed and expressed to obtain various forms of micromolecular genetic engineering antibodies, such as, fragment antigen-binding (Fab) antibody, F(ab)2, single chain antibody, Nanobody, antibody fusion protein, immunoglobulin G (IgG) complete antibody, etc., which can be used for preparing antibody drugs for diagnosing and treating tumor and / or inhibiting viscera fibrosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to an anti-recombinant basic fibroblast growth factor (or called fibroblast growth factor 2, FGF-2) human scFv antibody and application thereof. Background technique [0002] Basic fibroblast growth factor (bFGF), also known as fibroblast growth factor 2 (FGF-2), is a member of the fibroblast growth factor (FGF) family The earliest and most studied factor among the 23 known members. bFGF has a variety of biological effects, including growth-promoting, division, and differentiation functions; it has a strong pro-proliferation effect on fibroblasts. bFGF is a multifunctional molecule with multiple binding epitopes on the surface of the molecule, such as receptor binding sites, heparin binding sites, integrin binding sites and other epitopes. Antibodies against different epitopes have different functions. Moreover, monoclonal antibodies against the same antigen have a very la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13G01N33/577A61K39/395A61P13/12A61P11/00A61P1/16A61P35/00
Inventor 邓宁吕卫东黄静怡王宏宋其芳
Owner JINAN UNIVERSITY
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