Polypeptides specifically binding to CD56 molecules and application of polypeptides

A specific and molecular technology, applied in the field of biomedicine, can solve problems such as the unsatisfactory therapeutic effect of anti-CD56 antibody, enhance the killing effect of tumor cells, etc., and achieve the effect of enhancing anti-tumor effect, high application value, and small molecular weight

Active Publication Date: 2017-10-10
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the therapeutic effect of anti-CD56 antibody alone is not very satisfactory, and further studies have used anti-CD56 antibody as a targeted transport molecule, combined with radioisotopes, cytotoxic drugs, cyto...

Method used

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  • Polypeptides specifically binding to CD56 molecules and application of polypeptides
  • Polypeptides specifically binding to CD56 molecules and application of polypeptides
  • Polypeptides specifically binding to CD56 molecules and application of polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 T7 phage liver cancer cDNA library screening CD56 non-antibody binding protein

[0030] Using T7 phage display technology, the peptides that can specifically bind to CD56 were screened from the human liver cancer cDNA library, the peptides with strong binding ability were selected by ELISA, and the DNA sequence of the inserted phage was sequenced and analyzed to artificially synthesize the peptides.

[0031] 1.1 Preparation of medium

[0032] (1) Preparation of LB liquid medium (1L volume):

[0033] Element

weight

Bacto typtone

10g

Yeast extract

5g

NaCl

10g

[0034] Dissolve in secondary water, adjust the pH to 7.5 with 1N NaOH, and sterilize with high-pressure steam for 20 minutes.

[0035](2) Preparation of LB solid medium (1L volume):

[0036] Element

weight

Bacto typtone

10g

Yeast extract

5g

NaCl

10g

Agar

15g

[0037] Dissolve in secondary water, a...

Embodiment 2

[0083] Example 2. Specific research on the binding of CD56-nABP polypeptide to cells

[0084] 2.1 Specific research on the binding of CD56-nABP polypeptide to U2 OS cells and CTSC-2 cells

[0085] (1) The cover slip was sterilized with alcohol and placed in a 6-well plate, and the cover slip was coated with 0.1% rat tail type I collagen for 1 h at room temperature.

[0086] (2) After washing once with PBS, the cells were inoculated on the coverslip, and cultured in a 37°C incubator for 24 hours. After the cells adhered to the wall, peptides No. 7 and No. 8 were added and incubated for 30 minutes.

[0087] (3) Wash once more with PBS, add 4% PFA to fix for 15min.

[0088] (4) After washing 3 times with PBS, incubate with blocking solution (5% BSA+10% goat serum) for 1 h.

[0089] (5) Add diluted CD56 antibody (1:100) and incubate overnight at 4°C.

[0090] (6) Then wash with PBS for 3 times, add Anti-Mouse IgG fluorescent secondary antibody (1:1000), and incubate at room tem...

Embodiment 3

[0102] Example 3. Cytotoxicity experiment of CD56-nABP polypeptide

[0103] After the U2OS cells were digested and resuspended, the cells were counted, and 3000 cells were seeded in each well of a 96-well plate, and the cells were cultured with 100 μl (DF+10% FBS) medium, and placed in CO 2 Cultivate in an incubator for 24 hours. After the cells adhere to the wall, add different concentrations of polypeptides (0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml) , Cell viability was detected by CCK8 after 72h.

[0104] The result is as Figure 5 As shown, Figures A and B respectively show the toxic effects of No. 7 and No. 8 polypeptides on U2 OS cells. The results of statistical analysis showed that there was no significant difference in the cell viability of U2 OS cells cultured in the medium with different concentration gradients of the polypeptide and the medium without the addition of the polypeptide, indicating that the No. 7 and No. 8 polyp...

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Abstract

The invention provides polypeptides specifically binding to CD56 molecules (neural cell adhesion molecules) and an application of the polypeptides. The polypeptides are at least one selected from the group consisting of a No.7 polypeptide and a No.8 polypeptide, and the amino acid sequence of the No.7 polypeptide is shown as a sequence 1 in a sequence table and the amino acid sequence of the No.8 polypeptide is shown as a sequence 2 in the sequence table. The polypeptides provided by the invention can specifically bind to the CD56 molecules, and can be produced by an artificial synthetic method or a genetic engineering method. Compared with antibodies, the polypeptides provided by the invention have the following characteristics: the polypeptides are easy to prepare, and have a small molecular weight, an immunological rejection reaction is not easy to produce and the like; and the polypeptides have small toxic and side effects, and after binding to CD56 positive cells, the polypeptides do not have an obvious killing ability or proliferation inhibition effect. The polypeptides provided by the invention can be used as CD56 positive expression cell markers, and the polypeptides can also be used as substitutes for immunofluorescent CD56 antibodies and the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to polypeptides capable of specifically binding to CD56 molecules and applications thereof. Background technique [0002] CD56 molecule, also known as neural cell adhesion molecule (NCAM), is a member of the immunoglobulin superfamily. CD56 is a glycoprotein containing a polypeptide chain. The extracellular part of CD56 molecule includes 5 Ig-like domains and 2 type III fibronectin-like domains. Due to the different splicing methods of its mRNA, three different molecular subtypes are finally generated: 1.120kDa subtype, anchored on the cell membrane by GPI (glycosyl-phosphatidylinositol), mainly expressed in normal tissues and well-differentiated tissues, such as NK cells , nerve tissue, nerve-muscle junction, neuroendocrine tissue, endocrine tissue, etc. 2. The 140kDa and 180kDa subtypes contain a transmembrane domain and are mainly expressed on undifferentiated tissues or malignant tu...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N5/0783C12N5/079C12N5/0793C12N5/09A61K47/42A61P35/00
Inventor 张雁黄鸿兴赵龙汪华
Owner SUN YAT SEN UNIV
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