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Antigen and antibody of neural recognition molecule contactin 6 and application thereof

A technology for identifying molecules and antibodies, applied in the field of immunology, can solve problems such as lack of pertinence and specificity, lack of axon regeneration antibodies, lack of specificity and affinity of antibodies, etc.

Active Publication Date: 2017-08-29
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no antibody against axon regeneration in the field of spinal cord injury in China
In foreign countries, due to the lack of in-depth analysis of structural biology and bioinformatics when designing antibodies, antibodies often lack sufficient specificity and affinity, and the microenvironment formed by the conformation of antibody binding sites is difficult to achieve further development. Optimization; in addition, the functional blocking effect of some polyclonal antibodies lacks sufficient pertinence and specificity compared with monoclonal antibodies

Method used

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  • Antigen and antibody of neural recognition molecule contactin 6 and application thereof
  • Antigen and antibody of neural recognition molecule contactin 6 and application thereof
  • Antigen and antibody of neural recognition molecule contactin 6 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of the antigen of neural recognition molecule contactin 6

[0030] Using relevant bioinformatics software (MOE software, Bowtie software, BWA software, Clustalw, etc.), the relevant key sequences of the homologous proteins (contactin-4, contactin-5, contactin-6) of the contactin 6 protein were compared, among which , the key amino acid sequences of contactin-4, contactin-5, and contactin-6 are respectively shown in SEQ ID No.2, SEQ ID No.3, and SEQ ID No.4.

[0031] Using the known crystal structure of the contactin 6FN region, the crystal structure of the IgG region and the PTPR protein complex, in several aspects such as improving the stability of antigen binding, specific binding of the ideal CDR conformation, and conformational changes caused by FR folding, To analyze the effect of antibody design for blocking contactin 6 trans-homologous binding on antibody affinity and specificity.

[0032] Wherein, the corresponding sequence of the IgG 2-3 ...

Embodiment 2

[0038] Example 2 Preparation of antibody to neural recognition molecule contactin 6

[0039]Immunize mice with the above targetedly designed contactin 6 antigen polypeptide, select 6-8-week-old female wild-type mice, let them enter peripheral immune organs through blood circulation or lymphatic circulation, and stimulate corresponding B lymphocyte clones to differentiate into sensitized B lymphocytes. Then the mouse spleen was taken out to prepare a spleen cell suspension, and the myeloma cells and mouse spleen cells were mixed at a ratio of 1:2 to 1:5, and hybridoma cells were formed under the action of polyethylene glycol. In HAT medium, only fused hybridoma cells can obtain hypoxanthine-guanine phosphoribosyltransferase from splenocytes, and have the property of unlimited proliferation of myeloma cells, so they can survive and proliferate in HAT medium. The hybridoma cells were cloned and cultured by the limited dilution method, and the positive hybridoma cells capable of ...

Embodiment 3

[0040] Example 3 Screening and functional testing of antibodies

[0041] (1) Elisa detection

[0042] Spinal cord tissue collection: cut out 5 mm long tissue from the eighth to tenth segment of the thoracic spinal cord tissue centered on the injured area, homogenate the obtained spinal cord tissue, add the tissue to normal saline and mash it, and centrifuge at 3000 rpm for 10 minutes Then take the supernatant. Take out the required strips from the aluminum foil bag after equilibrating at room temperature for 20 minutes, and seal the remaining strips with a ziplock bag and store them at 4°C.

[0043] Set standard wells and sample wells, add 50 μl of IgG solution standard of known concentration to each standard well; add 10 μl of the sample to be tested in the sample well, and add 40 μl of diluent; do not add to the blank well. In addition to the blank wells, add 100 μl of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard wells and sample wel...

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Abstract

The invention relates to antigen of neural recognition molecule contactin 6, and an amino acid sequence of the antigen is shown in SEQ ID No.1. The invention also discloses an antibody prepared by the above antigen of neural recognition molecule contactin 6. The invention further discloses an application of the antibody in preparation of drugs for treating spinal cord injury. The antibody of the invention can effectively block the homologous association of contactin 6 and promote nerve axon growth, and the application of the antibody used in spinal cord injury can promote nerve axon regeneration and functional improvement.

Description

technical field [0001] The invention relates to the field of immunology, in particular to an antigen, antibody and application of a nerve recognition molecule contactin 6. Background technique [0002] In the process of functional recovery after spinal cord injury, the axon regeneration of the corticospinal tract controlling voluntary movement has been one of the research hotspots in the field of spinal cord injury. After spinal cord injury, scar tissue composed of reactive glial cells, microglia, fibroblasts, macrophages, and perivascular tunica cells constitutes a barrier that prevents axonal regeneration. Various growth inhibitory and axon-guiding molecules are induced accordingly and inhibit axon growth. Removal of inhibitors at the site of injury allowed surviving axons to re-sprout, but was not sufficient to cause axon regrowth. Studies have found that the intrinsic growth ability of neurons plays an important role in the regulation of axon regeneration, but how scar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K16/28A61K39/395A61P25/00
CPCA61K2039/505C07K14/70503C07K16/2803
Inventor 刘耀波黄振晖高亚荣孙玉慧刘洋王坚
Owner SUZHOU UNIV
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