A specific d-type polypeptide targeting lymphoma cell lines and its application

A cancer cell-targeted combination technology, applied in the field of biomedicine, can solve problems such as poor patient compliance, short half-life, and difficulty in popularization and application

Active Publication Date: 2021-09-10
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Compared with traditional chemically synthesized drugs, protein and peptide drugs have the advantages of clear mechanism of action, less dosage, and less toxic and side effects. However, due to the short half-life of these drugs in the body, repeated administration is required, resulting in poor patient compliance. Difficult to promote the application

Method used

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  • A specific d-type polypeptide targeting lymphoma cell lines and its application
  • A specific d-type polypeptide targeting lymphoma cell lines and its application
  • A specific d-type polypeptide targeting lymphoma cell lines and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Culture of Jeko-1 lymphoma cell line, phage titer determination, phage display panning

[0024] A. Culture of Jeko-1 lymphoma cell line:

[0025] The Jeko-1 lymphoma cell line (ATCC, American Type Culture Collection) was placed at 37°C, 5% CO 2 The cells were cultured in an incubator, and 10% calf serum, 100 U / mL penicillin and 100 U / mL streptomycin were added to the 1640 medium.

[0026] B. Phage titer determination:

[0027] (1) ER2738 glycerol bacteria (NEB Company) were taken at -80°C, streaked on LB-Tet plate, and cultured upside down at 37°C for 12h-16h.

[0028] (2) Use a sterile tip to pick a single colony of ER2738 into 5-10mL LB-Tet medium, culture it on a shaker at 37°C and 220rpm until mid-log phase (OD 600 = 0.5).

[0029] (3) Heat and melt the Top agar in a microwave oven, divide it into 3mL equal portions in sterile centrifuge tubes, and keep warm at 45°C for later use.

[0030] (4) Pre-warm the LB / IPTG / Xgal plate at 37°C.

[0031] (5) Th...

Embodiment 2

[0066] Example 2. Acquisition of phage monoclonal and analysis of biological information

[0067] Monoclonal phage amplification and purification:

[0068] (1) Take ER2738 glycerol bacteria at -80°C, streak on LB-Tet plate, and incubate upside down at 37°C for 12h-16h.

[0069] (2) The ER2738 monoclonal plaques obtained in step (1) were cultured in 20 mL LB-Tet at 37° C. and 220 rpm on a shaker for 12h-16h.

[0070] (3) Inoculate ER2738 overnight culture in step (2) at 1:100 dilution in LB medium, divide 1 mL into 15 mL centrifuge tubes.

[0071] (4) Take the LB plate used for the titer determination of the fourth round of panning, pick blue phage plaques with the tip of a pipette in step (3) 1mLER2738 bacterial solution, and culture on a shaker at 37°C and 220rpm for 4h-5h.

[0072] (5) Transfer the culture to a centrifuge tube, centrifuge at 14000rpm for 30s, transfer the supernatant to a new centrifuge tube, repeat the centrifugation for 30s, transfer 80% of the supernata...

Embodiment 3

[0088] Example 3. The cultivation of lymphoma cells, the extraction and primary culture of human lymphocytes, and the polypeptide immunofluorescence experiment

[0089] Culture of Lymphoma Cells:

[0090] Lymphoma cell lines Jeko-1, Romas, Raji, Granta-5, Su-4 were purchased from ATCC (American Type Culture Collection), and cultured in RPMI-1640 medium containing 10% fetal bovine serum.

[0091] Mouse mast cell carcinoma cell line P815 was purchased from ATCC (American Type Culture Collection), cultured in DMEM medium containing 10% fetal bovine serum, and all cells were maintained at 37°C, 5% CO 2 Routine culture in a cell incubator.

[0092] Extraction and primary culture of human lymphocytes:

[0093] (1) Transfer the blood of a normal person into a centrifuge tube, centrifuge at 500 g for 8 min, and separate the supernatant serum into a new centrifuge tube.

[0094] (2) Add an equal volume of PBS to the serum obtained in step (1), and mix well.

[0095] (3) Take a new ...

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PUM

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Abstract

The present invention relates to a specific polypeptide targeting and binding to lymphoma cells and its application, belonging to the field of biomedicine; the amino acid sequence of the polypeptide D-REVTUZG12 is: RMLSGHGANPHM; the polypeptide D-REVTUZG12 is composed of D-type amino acids; The invention proves that the D-type reverse sequence polypeptide D-REVTUZG12 has targeted binding activity to four lymphoma cell lines Jeko-1, P815, Raji and Su-4 through in vitro cell uptake and distribution experiments, but it has no effect on Romas and Granta-519 Lymphoma cell lines and human normal lymphocytes have no target binding activity; the polypeptide involved in the present invention has good target binding activity to multiple lymphoma cell lines, and has clinical significance in the diagnosis and targeted therapy of lymphoma. important application prospects.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a specific D-type polypeptide targeting and binding to lymphoma cell lines and its application. Background technique [0002] Malignant lymphoma is one of the ten most common tumors in my country. According to the data published in "The Lancet" magazine in 2018, the incidence of malignant lymphoma in China is about 5 / 100,000, and the five-year survival rate is only about 38.3%. Lymphoma is a kind of monoclonal proliferative malignant tumor originating from the lymphatic hematopoietic system. It is mainly divided into two categories: Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). The incidence rate is high. [0003] Mantle cell lymphoma (Mantle cell lymphoma, MCL) is a subtype of B-cell non-Hodgkin's lymphoma (B-NHL), accounting for about 6% of non-Hodgkin's lymphoma. MCL is considered an indolent and aggressive malignant lymphoma that usually begins with enlar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08G01N33/68G01N33/574A61K47/42A61P35/00
CPCA61K47/42A61P35/00C07K7/08G01N33/57407G01N33/68G01N2410/00
Inventor 刘晗青梁智全张雅菲屠志刚卢子文
Owner JIANGSU UNIV
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