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Novel antigen epitope based on CD271 and application thereof

An antigen epitope, a new type of technology, applied in the biological field, can solve the problems of binding influence, shedding, undetectable, etc., and achieve the effect of stable binding, strong affinity and low homology

Active Publication Date: 2021-05-28
苏州铂维生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after the expression of CD271, the CRD region will be hydrolyzed by protease and fall off, so the binding of antibodies targeting the CRD region to CD271-positive cells will be affected, and even the expression of CD271 cannot be detected in a certain stage of cell metabolism, so it is urgent New epitopes need to be developed to provide new tools for CD271-based antibody, vaccine research and related detection or disease treatment

Method used

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  • Novel antigen epitope based on CD271 and application thereof
  • Novel antigen epitope based on CD271 and application thereof
  • Novel antigen epitope based on CD271 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] [Example 1] Design, expression and purification of human CD271 antigen epitope peptide plasmid

[0063] 1. Selection of human CD271 epitope peptide

[0064] According to the full-length amino acid sequence of CD271 protein retrieved by NCBI, DNAStar software, IEDB website, etc. were used to analyze the antigenic determinant, and finally the epitope peptide was determined to be located in the SEQ ID NO.1 of the outer Stalk region of CD271 (named CD271T). Among them, a series of epitope peptides are designed to meet:

[0065] (1) located in the polypeptide chain SEQ ID NO.1; or,

[0066] (2) An amino acid sequence at least 80% identical to the sequence of SEQ ID NO. 1 in (1).

[0067] 2. Design of CD271T expression plasmid

[0068] According to the NCBI retrieval of the complete gene sequence of CD271 protein and the amino acid sequence of CD271T, the prokaryotic expression codon was optimized by Suzhou Synbio Co., Ltd., and cloned into the pET-28a(+) vector together w...

Embodiment 2

[0089] [Example 2] Proteolytic cleavage reaction and purification of human CD271T antigen peptide

[0090] 1. Protease digestion reaction and SDS-PAGE identification of human CD271T antigen peptide

[0091] (1) enzyme digestion system

[0092] The enzyme digestion system is shown in Table 1, reacted in 30°C water bath for 1h, 2h, 4h, 6h, and carried out SDS-PAGE identification.

[0093] Table 1

[0094]

[0095]

[0096] (2) SDS-PAGE identification

[0097] Take 20 μL samples before enzyme digestion and 1h, 2h, 4h, and 6h after enzyme digestion, add 5×reduced SDS-PAGE loading buffer, heat at 95°C for 10 minutes, and centrifuge at 12,000 rpm for 5 minutes. Take 10 μL of each prepared sample for SDS-PAGE gel electrophoresis detection. For specific results, see image 3 Figure A in .

[0098] 2. Human CD271 immune antigen purification

[0099] Expand the enzyme digestion system according to the above reaction system, react in a water bath at 20°C for 2 hours, filter t...

Embodiment 3

[0100] [Example 3] Using the antigenic peptide in Example 1 to screen antibodies

[0101] 1. Antigen Preparation

[0102] 1) Antigen preparation for priming: Take a 1 mL sterile EP tube, dilute 100 μg of the immunogen with PBS to 100 μL and put it in the EP tube; shake well Freund’s complete adjuvant to mix the precipitated bifidobacteria evenly; take 100 μL Freund’s complete adjuvant Add the complete adjuvant to a 5mL sterile EP tube, then add the diluted immunogen, so that the volume ratio of the antigen to the adjuvant is 1:1, put the centrifuge tube in the ice box; prepare the ultrasonic breaker, Place the centrifuge tube in an ice box for sonication. Ultrasound conditions are total power 200W, total time 10min, working time 5 seconds, interval time 6 seconds. Observe the emulsification of the solution in real time; inhale the emulsified antigen with a 1 ml disposable syringe, and place it in an ice box for later use.

[0103] 2) Antigen preparation for the second and t...

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Abstract

The invention relates to a novel antigen epitope peptide based on CD271 and application of the novel antigen epitope peptide. The antigen epitope peptide is: (1) located in SEQ ID NO.1; or (2) is located in an amino acid sequence having at least 80% identity with the SEQ ID NO.1 sequence in (1); wherein SEQ ID NO.1 contains one or more antigen epitopes, and the length of the amino acid sequence of the antigen epitope peptide is 5-70% of the full length of the SEQ ID NO.1. An antibody, a nucleic acid aptamer, a vaccine, nanoparticles and the like targeting the antigen peptide can be stably combined with natural cells with CD271 protein. The antigen epitope peptide or nucleic acid, fusion protein, carrier or host cell and the like containing the antigen epitope peptide have important application significance in the fields of preparation of CD271 related detection reagents, disease treatment biological drugs, vaccines and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel CD271 epitope peptide and its application. Background technique [0002] CD271 is a low-affinity nerve growth factor receptor (LNGFR), a member of the tumor necrosis factor receptor superfamily, with a relative molecular mass of 75kD, also known as p75NTR, which contains three regions: cysteine-rich Extramembrane region, transmembrane region and intracellular region composed of 155 amino acid residues. CD271 is not only expressed in the nervous system, it is closely related to the development, differentiation and survival of nerve cells, and can also identify tumor stem cells and mesenchymal stem cells. In the field of tumor treatment, CD271 is highly expressed on the surface of various tumor cells such as melanoma or tumor stem cells. Monoclonal antibodies targeting CD271 and their conjugated drugs can play a targeted killing role to treat tumors. In the field of mesenchym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K19/00C12N15/12C12N15/70C12N1/21C07K16/28A61K39/00A61P35/00A61P25/00A61P43/00C12R1/19
CPCC07K14/70578C12N15/70C07K16/2878A61K39/0005A61K39/001117A61P35/00A61P25/00A61P43/00C07K2319/35C07K2317/34C07K2317/76C07K2317/92C07K2317/565C07K2317/56A61K2039/53Y02A50/30
Inventor 路慧丽王紫嫣杨慧朱建伟
Owner 苏州铂维生物科技有限公司
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