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Polypeptide specifically binding to CD34 molecule and application thereof

A specific and molecular technology, applied in the field of biomedicine, can solve the problems of legacy medical safety hazards, large preparation workload, long production cycle, etc., and achieve the effect of reducing stem cell transplantation complications, good permeability and low cost.

Active Publication Date: 2016-01-20
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some problems in the preparation and clinical application of CD34 antibodies: first, because the immunogenicity of CD34 molecules is very weak, it is difficult to stimulate the body to produce B cells that secrete specific antibodies, resulting in a large workload and low success rate for the preparation of CD34 antibodies. It has the disadvantages of high cost and long production cycle; secondly, in the process of hematopoietic stem cell transplantation, these mouse-derived antibodies will inevitably enter the human body along with hematopoietic stem cells, thereby causing human anti-mouse antibody immune response (humananti-mouseantibodyresponse, HAMA ), leaving medical safety hazards

Method used

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  • Polypeptide specifically binding to CD34 molecule and application thereof
  • Polypeptide specifically binding to CD34 molecule and application thereof
  • Polypeptide specifically binding to CD34 molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1CD34

[0049] Example 1 The screening of CD34-binding phage

[0050] 1) Enrichment of CD34-binding phages with CD34 as the target molecule

[0051] The 96-well plate was coated with 5 μg / mL CD34 molecule (purchased from PeproTech), and the T7 phage lysate displaying random polypeptides in the T7 human cDNA library (Novagen) was mixed with 3% skimmed milk powder at a mass ratio of 1:1. , and incubated with CD34 molecule-coated 96-well plate for 30 minutes. Then, the non-specifically bound phages were eluted with washing solution, and the T7 phages bound to CD34 were enriched. After repeated screening, the recovery rate of the phage library was no longer increased. Petri dishes not coated with CD34 molecules were used as the control group.

[0052] The titer of phage input in the first round of screening was 6.4×10 10 cfu, the recovered phage titer was 5.4×10 5 cfu, the recovered phage was amplified, and the titer reached 4.2×10 9 cfu, continue to invest in the second round of scr...

Embodiment 2CD34

[0060] Example 2Co-immunoprecipitation of CD34 binding polypeptide

[0061] (1) Total protein extraction: Wash K562 cells once with pre-cooled PBS buffer, discard PBS, add 500 μL of non-denatured protein lysate containing protease inhibitors to the culture dish, and collect the cells into a 1.5 mL centrifuge tube , sonicated 4 times, 4 seconds each time, placed on ice for 30 minutes, centrifuged at 8000 rpm at 4°C for 20 minutes, and transferred the supernatant to a new centrifuge tube to obtain the total cell protein product;

[0062] (2) Pretreatment of total cell protein product: add 50 μL normal goat serum blocking solution to the total cell protein product, incubate on ice for 1 hour, then add 100 μL protein G microsphere suspension (Calbiochem), and incubate at 4°C for 20 minutes , centrifuging to remove protein G microspheres;

[0063] (3) Co-immunoprecipitation: Add CD34 antibody (AR) to the total protein pretreated in step (2) as the experimental group, and add the s...

Embodiment 3

[0068] Example 3 Binding of CD34-binding polypeptides to CD34 molecules of CD34-positive cells

[0069] (1) Cell preparation: place K562 cell suspension at 37°C, 5% CO 2 cultured in an incubator. When the cell density reaches 80%, centrifuge to discard the culture medium, wash once with PBS buffer, centrifuge, take the cell suspension, drop it on the glass slide coated with poly-lysine, fix it with cold acetone after drying for 10 minute.

[0070] (2) Wash with PBS buffer 3 times, 5 minutes each time. Then block with 5% BSA blocking solution for 1 hour at room temperature.

[0071] (3) Primary antibody incubation: the primary antibody dilution of CD34 antibody (AR) was dropped onto the glass slide, and incubated overnight at 4°C. Wash with PBS buffer 3 times, 5 minutes each time.

[0072] (4) Secondary antibody incubation and peptide binding: Peptides No. 17 and No. 19 were added to the diluted AlexaFluor561-labeled anti-rabbit antibody and mixed, then dropped onto the co...

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Abstract

The invention provides a polypeptide specifically binding to a CD34 molecule and application thereof. The polypeptide is at least one selected from the group consisting of polypeptide No. 17 and polypeptide No. 19, wherein the amino acid sequences of the polypeptide No. 17 and the polypeptide No. 19 are respectively shown in a sequence 1 and a sequence 2 in a sequence table. The polypeptide provided by the invention can specifically bind to the CD34 molecule and can be produced by using an artificial synthesis or genetic engineering method. Compared with an antibody, the polypeptide provided by the invention has the characteristics of easiness in preparation, small molecular weight, small possibility of incurrence of immunological rejection, etc.; moreover, the polypeptide has small toxic and side effect, can bind to CD34+ cells and does not exert obvious killing and propagation inhibiting effects on target cells. The polypeptide can be used as a marker for a cell with CD34 positive expression, or as a substitute for an immunofluorescence CD34 antibody, or the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to polypeptides capable of specifically binding to CD34 molecules and applications thereof. Background technique [0002] CD34 molecule is a highly glycosylated type I transmembrane protein with a molecular weight of 105-120kD, unique in structure, and specifically expressed in human and other mammalian hematopoietic stem / progenitor cells (hemopoietic stem / progenitor cells, HSC / HPC) surface, and as hematopoietic cells gradually mature, the expression of CD34 gradually decreases until it disappears. Secondly, CD34 molecule is also expressed in normal and tumor microvascular endothelial cells, and its mechanism of action in vascular endothelium is still unclear. [0003] Existing research results show that CD34 molecules play an important role in mediating intercellular adhesion, and participate in the transportation, localization and homing of HSC / HPC hematopoietic stem cells Binding of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K14/00A61K38/10A61K38/16A61K47/42A61P35/00A61P43/00G01N33/68G01N33/574C12N5/0789
CPCC07K7/08C07K14/00A61K38/00A61P31/00G01N2333/70596G01N33/5002G01N33/5434G01N33/68A61K38/04A61K2035/124G01N33/5008G01N33/54346G01N33/57426
Inventor 汪华赵龙张海霞张雁
Owner SUN YAT SEN UNIV
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