Single-domain antibody of heparin-binding epidermal growth factor and application thereof

An epidermal growth factor and single-domain antibody technology, applied in the field of biomedicine or biopharmaceuticals, can solve the problems of poor antibody stability, clinical treatment and detection limitations, and high production costs, achieving good stability, inhibiting migration and infiltration, and low cost. Effect

Active Publication Date: 2017-02-15
上海触研医学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the HB-EGF antibodies used in both clinical and scientific research ELISA kits are derived from traditional polyclonal and monoclonal antibodies, or human-mouse chimeric antibodies. These antibodies have poor stability and high production costs. They are not suitable for clinical treatment and detection. Restricted

Method used

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  • Single-domain antibody of heparin-binding epidermal growth factor and application thereof
  • Single-domain antibody of heparin-binding epidermal growth factor and application thereof
  • Single-domain antibody of heparin-binding epidermal growth factor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Screening of single domain antibodies against heparin-binding epidermal growth factor

[0045] (1) Coat 100 μg / mL of the partial protein encoding heparin-binding epidermal growth factor amino acid positions 63-149 (P6018, purchased from Yanofa Biotechnology Co., Ltd.) in Nunc immunotubes with PBS, 4°C Leave overnight.

[0046] (2) Add 5mL 5% skimmed milk the next day, and block at room temperature for 2 hours;

[0047] (3) After 2 hours, add 1 mL of single domain antibody phage display library (6001_hDAb, purchased from Source Biosciences, UK), and incubate at room temperature for 1 hour;

[0048] (4) Wash 20 times with PBST (PBS containing 0.05% Tween 20, SN330-2, purchased from Nanjing Shengxing Biotechnology Co., Ltd.);

[0049] (5) Add 100 μg / mL trypsin (T1426, purchased from sigma company) and incubate at room temperature for 1 hour. The added trypsin solution needs to fill the entire immunotube to obtain phages that specifically bind to HB-EGF and inf...

Embodiment 2

[0051] Embodiment 2: ELISA screening specificity single positive clone with phage

[0052] (1) From the phage clones enriched in Example 1, select 96 single colonies and inoculate them in TB medium containing 100 μg / mL ampicillin (1L TB medium contains 2.3g potassium dihydrogen phosphate, 12.52g Dipotassium hydrogen phosphate, 12g peptone, 24g yeast extract, 4mL glycerol), after the logarithmic growth phase, increase the concentration of 1mM IPTG, and culture overnight at 28°C;

[0053] (2) Transfer the culture supernatant to an ELISA plate coated with a part of the protein at amino acid positions 63-149 of heparin-binding epidermal growth factor (P6018, purchased from Yanofa Biotech Co., Ltd.) (this well is the sample well , the control well was added with PBS to replace the culture supernatant), and placed at room temperature for 1 hour;

[0054] (3) Wash off unbound antibody with PBST, add primary antibody mouse anti-myc 9E10 antibody (anti-myc mouse, purchased from Santa ...

Embodiment 3

[0059] Example 3: Expression and purification of single domain antibody in Escherichia coli

[0060] (1) Cloning the nucleotide sequence of the single-domain antibody obtained by sequencing analysis into the expression vector pET28a (purchased from LifeTechnology Company) to form a prokaryotic expression recombinant plasmid pET28a-Dab capable of expressing the single-domain antibody, and sequence the correct recombinant plasmid Transformed into expression bacteria BL21 (DE3) (purchased from Life Technology Company), spread on LB solid culture plate containing 100 μg / mL kanamycin, and cultivate overnight at 37°C;

[0061] (2) Select a single colony and inoculate it in 3 mL of LB culture medium containing kanamycin, and incubate overnight at 37°C;

[0062] (3) Inoculate 2 mL of overnight strain into 200 mL of LB medium, culture at 37°C until the OD value reaches 0.5, add IPTG, and culture overnight at 28°C;

[0063] (4) Collect the bacteria by centrifugation, crush the bacteria...

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Abstract

The invention relates to a single-domain antibody of a heparin-binding epidermal growth factor and the application thereof, and belongs to the field of the biological medicine or the biological pharmaceutical technology. According to the technical scheme of the invention, firstly, a strain of single-domain antibody that is high in affinity to HB-EGF is obtained based on the phage antibody library panning and sequencing technology, and then the single-domain antibody is obtained through the expression and purification process of an escherichia coli expression system. Meanwhile, the purified single-domain antibody is applied to the ELISA detection of the HB-EGF and the content detection of the HB-EGF in a sample. The single-domain antibody can inhibit the infiltration and the migration of ovarian cancer cells. The single-domain antibody is easy in mass production, lower in cost and more stable. Therefore, the single-domain antibody has important application value in ELISA and the inhibition of tumors.

Description

technical field [0001] The invention belongs to the technical field of biomedicine or biopharmaceuticals, and relates to a heparin-binding epidermal growth factor single-domain antibody and application thereof. Background technique [0002] Heparin-binding EGF-like growth factor (HB-EGF) is a 22KD, O-glycosylated protein, a member of the EGF family, on fibroblasts, smooth muscle cells and epithelial cells etc. have mitogenic effect. HB-EGF is involved in many physiological processes, and is also related to many pathological processes, such as wound healing, eyelid formation, embryo implantation, heart development; it is also related to many pathological processes, such as atherosclerosis, smooth muscle hyperplasia, brain injury and tumorigenesis . [0003] More and more evidence shows that the abnormal regulation of HB-EGF signaling pathway is related to the occurrence and development of various cancers, and the main mechanisms that cause cancer include promoting tumor gro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13C12N15/70C12N1/21G01N33/68G01N33/53A61K39/395A61P35/00C12R1/19
CPCC07K16/22C07K2317/70C07K2317/569C07K2317/565C07K2317/567
Inventor 吕鹏陈克平潘晔屠志刚林锋聂志超王沁芸廉彩霞阮玲玲
Owner 上海触研医学技术有限公司
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