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Single-domain antibodies against tumor stem cell marker protein CD133 and application thereof

A single-domain antibody, anti-tumor technology, used in anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, etc., to reduce production costs and molecular weight. Small, structurally stable effect

Inactive Publication Date: 2019-08-16
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there are no studies on single-domain antibodies against the cancer stem cell marker protein CD133

Method used

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  • Single-domain antibodies against tumor stem cell marker protein CD133 and application thereof
  • Single-domain antibodies against tumor stem cell marker protein CD133 and application thereof
  • Single-domain antibodies against tumor stem cell marker protein CD133 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 preparation of helper phage

[0030](1) In a petri dish containing TYE solid medium, spread the TG1 glycerol bacteria purchased from Beyond by the three-zone method, and then place the petri dish in a 37°C constant temperature incubator for 12 to 16 hours;

[0031] (2) Pick a single colony of TG1 grown on TYE solid medium, inoculate it in 5 mL of 2×TY liquid medium, place it on a constant temperature shaker at 37°C, and cultivate it at 250 rpm for 12 to 16 hours;

[0032] (3) Transfer the bacterial culture solution in step (2) to another tube of 5mL 2×TY liquid medium at a ratio of 1:100, place it on a constant temperature shaker at 37°C, and cultivate it at 250rpm until the OD600 of the bacterial solution is about 0.5;

[0033] (4) Dilute the helper phage KM13 with PBS (from 10 12 / mL~10 4 / mL);

[0034] (5) Take 200 μL of the bacterial solution in step (3), add 10 μL of the diluted helper phage KM13 to it, mix well and place in a 37°C water bath for 30 mi...

Embodiment 2

[0042] Example 2 Expression of a phage single domain antibody library

[0043] (1) Take an appropriate amount of phage library (Source Bioscience, product number: 6001_hDAb), add 500mL 2×TY liquid medium (the medium additionally contains 100μg / mL ampicillin, 4% glucose), and culture on a constant temperature shaker at 37°C at 250rpm until the bacteria OD600=0.5;

[0044] (2) Add 2×10 12 A helper phage KM13 to the bacterial solution obtained in step (1), mix well and place in a 37°C water bath for 30 minutes. The 500mL culture was divided into 50mL tubes, centrifuged at 3200g for 10 minutes, the supernatant was discarded, the pellet was resuspended, and transferred to an Erlenmeyer flask containing 500mL of 2×TY liquid medium (the medium additionally contained 0.1% Glucose, 100 μg / ml ampicillin, 50 μg / ml kanamycin). Place in a constant temperature shaker at 25°C and shake at 250rpm for 16-20 hours.

Embodiment 3

[0045] Example 3 Purification of phage single domain antibody library

[0046] (1) Subpackage the overnight culture in Example 2 into 50 mL per tube, and centrifuge at room temperature for 20 minutes at 3200 g;

[0047] (2) Take the centrifuged supernatant and transfer it to another clean 50mL centrifuge tube, add 20% polyethylene glycol solution in a volume ratio of 1:4 to each tube, and incubate on ice for 1 hour;

[0048] (3) Place the centrifuge tube in step (2) at 4°C and centrifuge at 3200 g for 30 minutes, discard the supernatant, resuspend the pellet with 5 mL of PBS, add 1 mL of 20% PEG solution to it, and incubate on ice for 10 minutes;

[0049] (4) Place step (3) at 4°C and centrifuge at 3200g for 30 minutes, discard the supernatant, resuspend the pellet with 1 mL of PBS, then centrifuge at 4°C and 3200g for 5 minutes, and filter the supernatant with a 0.45 μM filter to sterilize;

[0050] (5) Estimate the titer of the phage library by measuring the absorbance at 2...

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Abstract

The invention discloses single-domain antibodies against a tumor stem cell marker protein CD133 and an application thereof. Antibodies binding to a CD133 extracellular segment are screened from a human single-domain antibody library by a phage display technology, and finally, two single-domain antibodies, named as HW-145 and HW-314 respectively, are finally screened out. Genes of the two single-domain antibodies are amplified, sequenced and cloned into expression vectors, and are subjected to expression purification with a single-domain antibody prokaryotic system; then ELISA detection, MTT, cell apoptosis and Transwell invasion experiments are carried out, and results show that the two single-domain antibodies have strong binding ability with antigens, can effectively inhibit the proliferation and invasion of tumor cells and can induce apoptosis of the tumor cells. The antibodies provided by the invention are full humanized single-domain antibodies, cannot produce immunogenicity whenbeing applied in a human body and have the function of inhibiting the tumor cells in vitro. The two single-domain antibodies lay the foundation for the development of tumor body drugs in the later period.

Description

technical field [0001] The invention relates to the field of antibodies, in particular to a single-domain antibody against tumor stem cell marker protein CD133 and its application. Background technique [0002] The immortality, migration, and loss of contact inhibition of tumor cells make tumors one of the most difficult diseases to cure. At present, tumor treatment methods mainly include surgical resection, radiotherapy or chemotherapy, but the existence of a group of tumor stem cells with stem cell-like characteristics in tumors leads to a very high rate of tumor recurrence after treatment. In 2006, the American Association for Cancer Research defined cancer stem cells as: cells in tumors that have the ability to self-renew and produce heterogeneous tumor cells. Such cells can be dormant for a long time in the body, and have a variety of drug-resistant molecules and are insensitive to external physical and chemical factors that kill tumor cells, resulting in tumor recurre...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/2896C07K2317/569C07K2317/73
Inventor 魏星刘志芳
Owner JINAN UNIVERSITY
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