Binding protein of tumor stem cell marker molecule EpCAM and application of binding protein

A cancer stem cell and protein-binding technology, which is applied in the field of the binding protein of the tumor stem cell marker molecule EpCAM, can solve the problem of not being able to effectively kill tumor stem cells, and achieve the effects of easy transformation, reduced production costs, and accelerated application

Inactive Publication Date: 2019-02-15
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the main ways to treat tumors include surgical resection, radiotherap

Method used

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  • Binding protein of tumor stem cell marker molecule EpCAM and application of binding protein
  • Binding protein of tumor stem cell marker molecule EpCAM and application of binding protein
  • Binding protein of tumor stem cell marker molecule EpCAM and application of binding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 preparation of helper phage

[0055] (1) Line the TG1 Escherichia coli clone strain in three zones on a petri dish with TYE solid medium, and place the marked petri dish upside down in a 37°C incubator for about 14 hours.

[0056] (2) Pick a single colony of TG1 on the petri dish and inoculate it into 5 mL of 2×TY liquid medium, and culture overnight at 37°C and 250 rpm.

[0057] (3) Transfer the bacterial solution obtained in step (2) to another 5mL 2×TY liquid medium at a ratio of 1:100 by volume, and culture it at 37°C and 250rpm until the bacterial solution OD 600 About 0.5.

[0058] (4) The KM13 helper phage was serially diluted with PBS (10 12 pfu / mL~10 4 pfu / mL).

[0059] (5) Take 200 μL of the bacterial solution cultured in step (3), add 10 μL of the diluted KM13 helper phage, and place the mixture in a 37° C. water bath for 30 minutes to obtain mixture A.

[0060] (6) Mix low-melting point agarose and 2×TY liquid medium, heat until completely mel...

Embodiment 2

[0066] Example 2 Preparation of phage binding protein library

[0067] (1) Thaw the TG1 bacteria containing binding protein particles (i.e. human binding protein phage library, SourceBioscience, London, UK) on ice, add 500mL 2×TY liquid medium (containing 100μg / mL ampicillin, 4% (w / v) Glucose), cultivated to OD at 37°C, 250rpm 600 = 0.5.

[0068] (2) add the 2 * 10 that embodiment 1 prepares 12 Helper phages were incubated in a water bath at 37°C for 30 minutes, 500 mL of the culture was divided into 50 mL tubes, centrifuged at 3200 g for 10 minutes, the supernatant was discarded, and the pellet was resuspended in 500 mL of 2×TY medium (containing 0.1% (w / v) glucose, 100 μg / mL ampicillin and 50 μg / mL kanamycin). 25°C and 250rpm shaking culture for 16-20h to obtain the phage binding protein library.

Embodiment 3

[0069] Example 3 Purification of the phage binding protein library

[0070] (1) The phage binding protein library prepared in Example 2 was divided into 50 mL tubes, 10 tubes in total.

[0071] (2) Centrifuge the centrifuge tube at 3200g for 20min, transfer the supernatant to a clean container, add 20% PEG-NaCl solution equivalent to 1 / 5 of the volume of the supernatant, mix well and distribute into 50mL centrifuge tubes Incubate on ice for 1 h.

[0072] (3) Centrifuge the centrifuge tube at 4°C, 3200g for 30min, discard the supernatant, resuspend the pellet with 5mL of PBS buffer, add 1mL of 20% PEG-NaCl solution to it, and incubate on ice for 10min.

[0073] (4) Centrifuge at 3200g at 4°C for 30min, discard the supernatant, resuspend the pellet with 1mL of PBS buffer, centrifuge at 3200g at 4°C for 5min, and filter the supernatant with a 0.45μM filter to sterilize.

[0074] (5) Roughly estimate the prepared phage titer by measuring the light absorption value at 260 nm: Dil...

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Abstract

The invention discloses a binding protein of a tumor stem cell marker molecule EpCAM and application of the binding protein. The binding protein is a binding protein formed by one or a combination ofat least two of an NB85 binding protein, an NB143 binding protein, an NB278 binding protein, an NB431 binding protein and an NB589 binding protein. The five binding proteins have the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 respectively. The invention also discloses the application of the binding protein of the tumor stem cell marker molecule EpCAM in preparation of antitumor drugs. The binding protein provided by the invention is high in structural stability, easy to modify and human-derived, has no immunogenicity in a human body, andcan be better applied to the development of the antitumor drugs.

Description

technical field [0001] The invention belongs to the field of binding proteins, in particular to a binding protein of tumor stem cell marker molecule EpCAM and application thereof. Background technique [0002] Studies have shown that there is heterogeneity among tumor cells, and tumor cells can form a hierarchical structure in tumor entities, in which there is a group of cells with the ability to initiate tumors, namely tumor stem cells. The theory of cancer stem cells holds that cancer stem cells are similar to ordinary tumor cells and have the characteristics of self-renewal, unlimited proliferation, pluripotency and drug resistance. At present, the main ways to treat tumors include surgical resection, radiotherapy or chemotherapy, but none of these methods can effectively kill tumor stem cells. Since cancer stem cells have the ability to initiate tumors, using genetic engineering technology to design specific biomarker molecules targeting cancer stem cells has become ano...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12N15/13C12N15/63A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/30C07K2317/76
Inventor 魏星刘志芳张伟雄戴琳
Owner JINAN UNIVERSITY
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