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Specific polypeptide targeted binding lymphoma cell lines and application thereof

A technology of targeted binding and cancer cells, applied in the field of biomedicine, can solve the problems of poor marker specificity and increase the economic burden of patients, and achieve the effect of enhancing the inhibitory effect and improving the killing effect.

Active Publication Date: 2018-11-16
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These markers have been widely used clinically, but due to factors such as different subtypes and poor marker specificity, it is often necessary to use multiple markers in combination
These markers have both advantages and limitations, and the combined use of multiple markers will inevitably increase the economic burden of patients

Method used

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  • Specific polypeptide targeted binding lymphoma cell lines and application thereof
  • Specific polypeptide targeted binding lymphoma cell lines and application thereof
  • Specific polypeptide targeted binding lymphoma cell lines and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Culture of Jeko-1 lymphoma cell line, determination of phage titer, phage display panning

[0026] A. Culture of Jeko-1 lymphoma cell line:

[0027] Jeko-1 lymphoma cell line (ATCC, American Type Culture Collection) placed at 37°C, 5% CO 2 For cell culture incubator, add 10% calf serum, 100U / mL penicillin and 100U / mL streptomycin to 1640 medium.

[0028] B. Determination of phage titer:

[0029] (1) Take ER2738 glycerol bacteria (NEB) at -80°C, streak it on an LB-Tet plate, and invert it at 37°C for 12h-16h.

[0030] (2) Use a sterile pipette tip to pick up a single colony of ER2738 in 5-10mL LB-Tet medium, and cultivate to mid-log phase (OD 600 =0.5).

[0031] (3) Heat and melt Top agar in a microwave oven, divide it into 3 mL aliquots and place them in sterile centrifuge tubes, and keep them warm at 45°C for later use.

[0032] (4) Pre-warm the LB / IPTG / Xgal plate at 37°C.

[0033] (5) Dilute the M13 phage (NEB company) sample with LB-Tet gradient, and replace the pipett...

Embodiment 2

[0068] Example 2. Acquisition of phage single clone and analysis of biological information

[0069] Monoclonal phage amplification and purification:

[0070] (1) Take ER2738 glycerol bacteria at -80℃, streak on LB-Tet plate, and invert at 37℃ for 12h-16h.

[0071] (2) Take the ER2738 monoclonal plaque obtained in step (1) and culture it in 20mL LB-Tet at 37°C on a 220rpm shaker for 12h-16h.

[0072] (3) Dilute the overnight culture of ER2738 in step (2) into LB medium at a dilution of 1:100, and divide 1 mL into 15 mL centrifuge tubes.

[0073] (4) Take the LB plate used for the titer determination of the fourth round of panning, pick the blue plaque with the pipette tip and place it in step (3) 1mlLER2738 bacterial solution, and incubate at 37°C for 4h-5h on a 220rpm shaker.

[0074] (5) Transfer the culture to a centrifuge tube, centrifuge at 14000 rpm for 30 seconds, transfer the supernatant to a new centrifuge tube, repeat the centrifugation for 30 seconds, transfer 80% of the supern...

Embodiment 3

[0094] Example 3. Lymphoma cell culture, human lymphocyte extraction and primary culture, fluorescent peptide synthesis, peptide immunofluorescence experiment

[0095] Culture of lymphoma cells:

[0096] Lymphoma cell lines Jeko-1, Romas, Raji, Su-4, Granta-519 (all purchased from ATCC, American Type Culture Collection) were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Mouse mast cell cancer cells P815 (purchased to ATCC, American Type Culture Collection) were cultured in DMEM medium containing 10% fetal bovine serum. All cells are at 37℃, 5% CO 2 The cell incubator is routinely cultured.

[0097] Extraction and primary culture of human lymphocytes:

[0098] (1) Transfer normal human blood into a 15mL centrifuge tube, centrifuge at 500g for 8min, and separate the supernatant serum into a new 15mL centrifuge tube.

[0099] (2) Add an equal volume of PBS with the serum obtained in step (1) to the serum and mix well.

[0100] (3) Take a new 15mL centrifuge tube, add the...

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Abstract

The invention relates to a specific polypeptide targeted binding to lymphoma cell lines and application thereof, and belongs to the field of biomedicine. The specific polypeptide is based on a phage display technology in vitro, in vitro panning and obtaining a specific polypeptide TUZG12 capable of targeted binding the lymphoma cell lines. In vitro cell uptake and distribution experiments prove that the polypeptide has targeted binding activities to the lymphoma cell lines. According to the specific polypeptide targeted binding to the lymphoma cell lines, the results of in vitro cell viabilityexperiments show that the pro-apoptotic peptide DKK can enhance the inhibitory effect of DKK on proliferation of lymphoma cells to a certain extent after coupling TUZG12 polypeptide. The polypeptidescreened in the invention has good targeted binding activities to multiple lymphoma cell lines, and can enhance the killing effect of cytotoxic polypeptides on target tumor cells, and has an importantapplication value in the clinical diagnosis and treatment of lymphoma.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a specific polypeptide targeted to bind to lymphoma cancer cell lines and its application. Background technique [0002] Compared with traditional antibody-based targeting molecules, small peptides that specifically bind to tumors have lower immunogenicity, lower production costs, and higher surface density (the number of peptides per unit surface is larger, so This conjugate has higher affinity) and better tissue penetration effect, can be used as a marker for tumor diagnosis, and can be coupled with traditional chemotherapy drugs to achieve targeted tumor therapy. Among the peptide drugs approved by the FDA, some peptide drugs have been designated for cancer treatment. Leuprolide is a gonadotropin releasing hormone analogue, which has been approved for the treatment of prostate cancer. The proteosome inhibitor bortezomib is used in the clinical treatment of multiple myeloma. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K19/00A61K47/42A61K38/16A61P35/00
CPCA61K38/00A61K47/42A61P35/00C07K7/08C07K2319/00
Inventor 刘晗青张雅菲卢子文屠志刚梁智全
Owner JIANGSU UNIV
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