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Polypeptide specifically binding to ATRA-resistant acute myeloid leukemia cells and preparation method and application thereof

A specific, cellular technology, applied in the field of biomedicine, can solve the problems of low washing efficiency, poor repeatability of screening results, and inability to completely remove non-specifically bound phages, and achieve the effects of increased probability, sufficient contact and sufficient binding.

Inactive Publication Date: 2018-12-18
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

By performing several rounds of "adsorption-elution-amplification" affinity screening and enrichment process between the library and the target cells, the phage clones displaying specific binding polypeptides to the target cells can be screened out from the library, but the traditional There are technical deficiencies in phage display random peptide library screening technology, among which the low washing efficiency makes it impossible to completely remove non-specifically bound phages, and makes the reproducibility of screening results poor

Method used

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  • Polypeptide specifically binding to ATRA-resistant acute myeloid leukemia cells and preparation method and application thereof
  • Polypeptide specifically binding to ATRA-resistant acute myeloid leukemia cells and preparation method and application thereof
  • Polypeptide specifically binding to ATRA-resistant acute myeloid leukemia cells and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Construction and identification of all-trans retinoic acid (ATRA) drug-resistant strains of acute myeloid leukemia cells

[0066] In this example, the following methods and steps are used to construct and identify all-trans retinoic acid-resistant strains of acute promyelocytic leukemia cells

[0067] 1. Concentration-increasing intermittent induction method to induce ATRA drug-resistant cell lines

[0068] 1. Take HL-60 cells in the logarithmic growth phase and adjust the cell concentration to 1×10 4 individual / mL.

[0069] 2. Add ATRA to the 1640 culture medium to make the final concentration 10 -9 mol / L, after culturing for 48 hours, the ATRA-containing medium was discarded, and the cells were cultured with RPMI-1640 medium for 2 to 3 generations.

[0070] 3. Culture the cells for 48 hours in 1640 culture medium containing ATRA, in which the concentration of ATRA is doubled, that is, 2×10 -9 mol / L.

[0071] 4. Repeated medium change and subculture, gradually inc...

Embodiment 2

[0088] Screening and preparation of phage-peptide (pepNo.5R5)

[0089] In this example, pepNo.5R5 was screened and prepared by the following methods and steps.

[0090] 1. Preliminary screening of phage clones that bind to ATRA-resistant AML cell lines

[0091] Using microfluidics-based phage display peptide library screening technology, multiple rounds of whole-cell subtractive screening were carried out to preliminarily screen out phage clones that specifically bind to the ATRA-resistant AML cell line HL-60R. The specific steps are as follows:

[0092] 1. Determination of phage titer

[0093] (1) Inoculate a single colony in LB-Tet medium and culture on a shaker at 37°C until mid-logarithmic growth (OD600≈0.5).

[0094] (2) When the bacteria grow, melt the high-layer agar in a water bath, and use one tube for each phage dilution. Store at 55°C for later use.

[0095] (3) Pre-warm the LB / IPTG / X-gal plate at 37°C, and prepare one plate for each phage dilution.

[0096](4)...

Embodiment 3

[0142] Binding specificity of pepNo.5R5 to drug-resistant cells

[0143] 1. Identify the binding of phage clones to various cell lines by titer assay

[0144] In this experiment, a phage clone carrying a pepNo.5R5 fragment was selected, and the binding ability of the phage clone to different cell lines was identified by a titer method, and the method was the same as in Example 2.

[0145] Experimental results such as image 3 As shown, the binding ability of pepNo.5R5 to the drug-resistant cell HL-60R was significantly stronger than that of the corresponding parental cell; with other normal cell lines (HL-7702, GES-1, 293T) and tumor cell lines (MCF-7, THP -1, HepG2, PC3) had very weak binding abilities. In addition, the 5R5 phage-displayed polypeptide can also bind to the multidrug-resistant strain CEM / C1 of acute lymphoblastic leukemia cells, indicating that this sequence may be a broad-spectrum binding peptide for drug-resistant cells.

[0146] 2. Immunofluorescence meth...

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Abstract

The invention discloses a polypeptide specifically binding to ATRA-resistant acute myeloid leukemia cells and a preparation method and application thereof. The polypeptide specifically binding to ATRA-resistant acute myeloid leukemia cells is named as pepNo. 5R5 and has a base sequence shown in the formula of SEQ ID NO. 1 and an amino acid sequence shown in the formula of SEQ ID NO. 2. The polypeptide fills a gap in the current lack of polypeptides that bind to ATRA drug-resistant AML cells. The screened polypeptide is a small molecule polypeptide which can be synthesized by an artificial method, has small molecular weight, high activity, strong penetrating power, high affinity, high specificity and low toxicity, and is suitable as a carrier for targeted therapy. The polypeptide has good tumor targeting effects and lays a solid foundation for the preclinical deep research of the polypeptide.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a polypeptide specifically combined with ATRA-resistant acute myeloid leukemia cells and its preparation method and application. Background technique [0002] Acute myeloid leukemia (AML) is the most common hematological malignancy in adults. Chemotherapy is currently the most important treatment and the basis of hematopoietic stem cell transplantation. But most patients still eventually die due to treatment failure. Even for the most successfully treated acute promyelocytic leukemia (APL), the optimized regimen of ATRA combined with anthracyclines or arsenic trioxide (ATO) can achieve 90% initial complete remission, but still 20-30% of patients Drug-resistant reaction to all-trans retinoic acid (ATRA), the three-year overall survival rate is only about 50%. There are many reasons for treatment failure, among which multidrug resistance is one of the most important reason...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N5/09A61K38/10A61P35/02
CPCA61K38/00A61P35/02C07K7/08C12N5/0694
Inventor 缪凤琴张建琼张莹张亚芬叶梦滢
Owner SOUTHEAST UNIV
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