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Clec9a-targeting affinity peptide WH peptide

An affinity and targeted technology, applied in peptides, medical preparations with non-active ingredients, and medical preparations containing active ingredients, etc., can solve the problems of poor antigen permeability and large molecular weight, and achieve strong antigenicity and obvious penetration. The effect of high permeability and high permeability

Active Publication Date: 2016-09-07
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Studies have shown that antigen targeting Clec9a can trigger a comprehensive immune response, so Clec9a antibodies and antigens are usually used for chemical coupling, but due to the large molecular weight of antibody-antigen complexes, there are many disadvantages, such as antigen penetration In addition, mouse anti-hClec9a mAb may cause great immunogenicity when applied to humans

Method used

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  • Clec9a-targeting affinity peptide WH peptide
  • Clec9a-targeting affinity peptide WH peptide
  • Clec9a-targeting affinity peptide WH peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Since the corresponding protein needs to be used as the basis before the screening of the phage display peptide library, the inventors prepared the mClec9a-CTLD protein as the screening premise, and the related preparation process is briefly introduced as follows.

[0068]First, transfect HEK-293T cells with the p3×FLAG-mClec9a-CTLD plasmid by the calcium phosphate transfection method. After transfection, culture in DMEM medium containing 0.3% FBS. The supernatant contains the expressed mClec9a-CTLD protein;

[0069] Second, Western Blotting was used to detect the mClec9a-CTLD protein expressed in the supernatant to ensure that the supernatant contained the target protein; the supernatants cultured for 40h and 64h after transfection were detected respectively, and the results were as follows figure 1 As shown, it can be seen from the figure that mClec9a-CTLD protein is expressed in the supernatant. In addition, the Western blotting verification results show that the mol...

Embodiment 2

[0074] Based on the mClec9a-CTLD protein prepared in Example 1, the solid-phase phage display 12-peptide library was used to screen for affinity phages with high affinity targeting the mClec9a-CTLD protein. After five rounds of panning, the phages were enriched. The phages eluted in the fifth round were randomly selected for sequencing, and one affinity phage with repeated sequences was obtained, which was named as phage WH. The screening process is carried out according to the NEB standard process, and the relevant process is briefly introduced as follows.

[0075] (1) Solid phase affinity panning process

[0076] The mClec9a-CTLD protein prepared in Example 1 was dissolved in 0.1M NaHCO3 (pH8.6) coating solution, coated in the wells of a 96-well plate, and incubated overnight at 4°C in a closed humid box; then the coating solution Discard completely, add 0.1M NaHCO3 (pH8.6) blocking solution containing 5% BSA, and incubate in a wet box at 4°C for 3h; after washing with TBST...

Embodiment 3

[0091] The present invention mainly introduces the artificial preparation process of the WH polypeptide obtained by screening in Example 2 using the Fmoc solid-phase synthesis method.

[0092] When preparing WH peptide by Fmoc solid-phase synthesis method, first swell the resin, wash and deprotect, then condense the first Fmoc-amino acid and HoBt, measure the substitution value after washing, then seal the head, deprotect and wash, indene detection Confirm that the deprotection is complete, then condense the second amino acid, and repeat the above steps until the synthesis of the polypeptide chain is completed; finally, after the synthetic polypeptide chain is cut and rotary evaporated, it is precipitated with ether to obtain the crude peptide, and then further washed with ice ether and centrifuged The crude peptide is further purified by precipitation, and finally the WH polypeptide product with higher purity is obtained. The detailed preparation process is described as follo...

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Abstract

The invention belongs to the technical field of tumor prevention and treatment and concretely relates to a Clec9a-targeting affinity peptide WH peptide and its preparation method and use. The peptide comprises 12 amino acids, has an amino acid sequence of WPRFHSSVFHTH and can be prepared through an Fmoc solid-phase synthesis method. The peptide can be used in tumor prevention and treatment. The peptide can be obtained through phage-displayed peptide library technology screening. The result of the research on an in-vivo and in-vitro foreign antigen cross presentation capacity and induction cases of specific CTL reaction shows that the WH peptide as a targeting carrier can well induce specific CTL response, improve IFN-gamma and related cell killing molecule expression quantity and improve tumor resistance. Through a computer simulated molecular docking technology, a possible docking area between the WH peptide and the Clec9a protein and action sites of the possible docking area can be researched. The peptide provides good reference and reference significance for related research.

Description

technical field [0001] The invention belongs to the technical field of tumor prevention and treatment, and specifically relates to an affinity peptide WH peptide targeting mClec9a, its preparation and application. Background technique [0002] Dendritic cells (DCs) are professional antigen-presenting cells that mediate T cell and B cell immune responses. Compared with other antigen-presenting cells (APCs), DCs have a superior ability to capture, process and present antigens, and only DCs can activate naïve T cells (T cells). DC induces a strong and specific anti-tumor immune response, so immunotherapy against DC is a very effective immunotherapy strategy. [0003] There are at least three types of DC in mice and humans: plasmacytoid DC (pDC), blood-derived conventional DC (cDC) and tissue-derived DC (tissue-derivedcDC). These three types of DCs can be divided into different DC subpopulations according to the surface molecules, functions and sources of the DC membrane surfa...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K39/385A61K47/48A61P35/00
CPCA61K39/385A61K2039/575A61K2039/6031C07K7/08
Inventor 高艳锋闫中义祁元明李国栋曹文鹏
Owner ZHENGZHOU UNIV
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