CN107098960B - Antigen and antibody of neural recognition molecule contact 6 and application - Google Patents

Abstract

The invention relates to an antigen of a nerve recognition molecule contact 6, and the amino acid sequence of the antigen is shown as SEQ ID No. 1. The invention also provides an antibody of the neural recognition molecule contact 6 prepared by adopting the antigen. The invention further discloses application of the antibody in preparing a medicine for treating spinal cord injury. The antibody of the invention can effectively block homologous binding of the contact 6 and promote the growth of the neurite, and the antibody can promote the regeneration and the functional improvement of the neurite when used in spinal cord injury.

Description

Antigen and antibody of neural recognition molecule contact 6 and application

Technical Field

The invention relates to the field of immunology, in particular to an antigen and an antibody of a neural recognition molecule contact 6 and application thereof.

Background

In the process of functional recovery after spinal cord injury, axonal regeneration of corticospinal tracts which control voluntary movement has been one of the research hotspots in the field of spinal cord injury. After spinal cord injury, scar tissue, consisting of reactive glial cells, microglia, fibroblasts, macrophages, and perivascular membrane cells, etc., constitute barriers to axon regeneration. Various growth inhibitory molecules, as well as axon-guiding molecules, will be induced and inhibit axon growth accordingly. Removal of the inhibitory factor from the lesion can cause surviving axons to re-bud, but not sufficiently to cause axon regeneration. It has been found that the intrinsic growth ability of neurons plays an important role in regulating axon regeneration, but how scar-forming cells regulate the intrinsic growth ability of neurons remains to be studied.

The contact family, as a sub-family of neural recognition molecules in the immunoglobulin superfamily, is specifically expressed in the nervous system, and six members are discovered so far: f3/contitin, TAG-1, BIG-2, NB-2 and contitin 6. The trans-homologous interaction of inducibly expressed contin 6 on damaged axons and inducibly expressed contin 6 on reactive astrocytes will transmit signals from reactive glial cells to neurons, resulting in contact inhibition of axon regeneration. At present, no antibody for promoting axon regeneration aiming at the protein of the contact 6 is used as clinical application.

Currently, relevant technologies to promote axon regeneration include antibody design patents directed to Nogo, and design patents directed to the enzymes of PTPs degraded by CSPGs. In the former, when the antibody is used for physical examination, the phenomenon of promoting axon regeneration can be observed in the fields of spinal cord injury and cerebral ischemia, but when the specific Nogo-NgR and a downstream signal transduction mechanism thereof are elucidated by using a transgenic mouse, strong controversy exists about the regeneration promoting effect of the Nogo-NgR. However, there are many classes of enzymes in the PTP family, but only a few classes (such as PTP sigma) have a certain effect on promoting axon regeneration, but the specific mechanism still needs to be further elucidated. In any of the related techniques, affinity and specificity of an antibody or an enzyme are improved by optimizing sequence design by means of phage screening, base back mutation, and the like which have been rapidly developed in recent years, and recognition of a key action site and functional detection and confirmation are improved, so that an optimal product can be obtained.

At present, no antibody aiming at axon regeneration exists in the field of spinal cord injury in China. In foreign countries, the prior antibody is not deeply analyzed in aspects of structural biology, bioinformatics and the like during design, so that the antibody often lacks sufficient specificity and affinity, and the microenvironment formed by the conformation of the antibody binding site is difficult to realize further optimization; in addition, the functional blocking effect of some polyclonal antibodies, relative to monoclonal antibodies, is less than adequate for targeting and specificity.

Disclosure of Invention

In order to solve the above-mentioned technical problems, it is an object of the present invention to provide an antigen and an antibody of a nerve recognition molecule, contact 6, and use thereof, wherein the antibody of the present invention can effectively block homologous binding of contact 6 and promote growth of neurites, and the use of the antibody in spinal cord injury can promote regeneration and functional improvement of neurites.

In one aspect, the invention provides an antigen of the neural recognition molecule contact 6, and the amino acid sequence of the antigen is shown as SEQ ID No. 1.

In still another aspect, the present invention also provides an antibody of the neural recognition molecule contact 6, wherein the antibody is prepared from the antigen.

Further, the antibody is a monoclonal antibody.

Further, the preparation method of the antibody comprises the following steps:

(1) immunizing a mouse by using the contictin 6 antigen, and stimulating the B lymphocyte of the mouse to clone and differentiate into sensitized B lymphocyte;

(2) extracting splenocytes of the mice, and mixing myeloma cells with the splenocytes to form hybridoma cells;

(3) culturing hybridoma cells in HAT culture medium by limiting dilution method, and screening out positive hybridoma cells;

(4) and proliferating the positive hybridoma cells in the mouse to obtain the antibody.

In another aspect, the invention also provides the application of the antibody in preparing a medicine for treating spinal cord injury.

By the scheme, the invention at least has the following advantages:

the invention utilizes the concerned research base of the structure biology of the contact family represented by the contact 6 protein, and combines with the bioinformatics related software such as MOE, etc., respectively in several aspects of antigen combination, ideal CDR conformation, FR folding, etc., designs and retains the key CDR area of the mouse monoclonal antibody, further improves the specificity and affinity of the antibody, and improves the microenvironment formed by the optimal conformation of the antibody combining area, thereby obtaining an antibody aiming at the contact signal path, the antibody can specifically promote the axon injury regeneration of the cortical spinal cord tract, and improves the repair capability after spinal cord injury, thereby providing beneficial clues for the rehabilitation in the field of spinal cord injury.

The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings.

Drawings

FIG. 1 is a crystal structure of a conjugate 6 protein IgG 2-3 domain complexed with a PTP family protein;

FIG. 2 is the crystal structure of the protein FN domain of contitin 6;

FIG. 3 is a diagram showing binding of the protein of contictin 6 to the key amino acid sites of the PTP family protein;

FIG. 4 is an alignment of key amino acids at the omega loop for the protein of contact 6 and for homologous proteins of the contact family;

FIG. 5 is a schematic representation of the contictin 6 protein amino acid modification sites;

FIG. 6 shows the binding potency of antibodies detected by Elisa assay;

FIG. 7 shows the detection of the binding ability of the antibody by Western Blot.

FIG. 8 is a graph showing the effect of antibodies in promoting the growth of protein-blocked axons measured in vitro by tissue culture techniques;

fig. 9 shows the results of the implantable osmotic pump technology in vivo detection of antibodies promoting mouse axon regeneration.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 preparation of antigen of neural recognition molecule contitin 6

The related key sequences of homologous proteins (contictin-4, contictin-5 and contictin-6) of the contictin 6 protein were aligned using bioinformatics related software (MOE software, Bowtie software, BWA software, Clustalw, etc.), wherein the key amino acid sequences of contitin-4, contitin-5 and contitin-6 were shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, respectively.

The influence of antibody design against blocking the reverse homologous binding of contact 6 on the affinity and specificity of the antibody was analyzed in several aspects, such as improvement in stability of antigen binding, specific binding of an ideal CDR conformation, conformational change due to FR folding, and the like, using the crystal structure of the FN region of contact 6, IgG region, and crystal structure of the PTPR protein complex, which are known separately.

Wherein, the corresponding sequence of IgG 2-3 structural domain of the protein of the contact 6 is shown as SEQ ID No. 5. This sequence is capable of forming a complex with a PTP family protein, whose crystal structure is shown in FIG. 1, and candidate regions of the antigen are compared according to the corresponding sequence and conformation at the time of design.

The corresponding sequence of the protein FN structural domain of the contact 6 is shown as SEQ ID No.6, the crystal structure is shown as figure 2, and the candidate regions of the antigen are compared according to the corresponding sequence and conformation during design.

The binding key amino acid sites of the protein of contact 6 and the protein of PTP family are shown in FIG. 3, from which the corresponding specific amino acid sequence and conformation of the binding segment can be seen, and the candidate regions of the antigen can be compared with reference to their binding properties and possible conformational changes caused during design.

FIG. 4 is a drawing showing the local alignment of key amino acids at the Ω -loop for a protein of contact 6 and a homologous protein of the cntn family (contact 5 protein), and FIGS. 4A and 4B are respectively the front and back views of the conformation of the key region of amino acids at the Ω -loop, showing the spatial conformation of contact 6 in this region compared with the homologous proteins of contact 4 and contact 5.

FIG. 5 shows the protein amino acid modification sites of contictin 6, and FIG. 5 lists all the modification sites of contictin 6, with reference to the specific influence on the corresponding sequence sites.

According to the method, the antigen of the neural recognition molecule contact 6 is designed, and the sequence of the antigen is shown as SEQ ID No. 1.

Example 2 preparation of an antibody against the neural recognition molecule contitin 6

The mice are immunized by the pertinently designed contactin 6 antigen polypeptide, and 6-8 weeks old female wild-type mice are selected to enter peripheral immune organs through blood circulation or lymphatic circulation, so that corresponding B lymphocytes are stimulated to clone and differentiate into sensitized B lymphocytes. Then taking out the spleen of the mouse, preparing spleen cell suspension, mixing myeloma cells and the spleen cells of the mouse according to the ratio of 1:2 to 1:5, and forming hybridoma cells under the action of polyethylene glycol. In HAT medium, only the fused hybridoma cells can obtain hypoxanthine guanine phosphoribosyl transferase from spleen cells, and have the property that myeloma cells can proliferate indefinitely, thereby being able to survive and proliferate in HAT medium. And (3) carrying out cloning culture on the hybridoma cells by adopting a limiting dilution method, and screening out positive hybridoma cells capable of producing the required monoclonal antibody. The hybridoma cells are proliferated in the abdominal cavity of the mouse, and the mouse can produce and secrete the monoclonal antibody in the abdominal cavity. After about 1-2 weeks, the abdomen of the mice expanded. The ascites is extracted by a syringe to obtain a plurality of monoclonal antibodies which are respectively named aCNTN6-001, aCNTN6-002, aCNTN6-003 and the like.

Example 3 screening and functional testing of antibodies

(1) Elisa test

Taking spinal cord tissues: cutting tissue with length of 5mm from the eighth to tenth section of thoracic section of spinal cord tissue with the damaged area as center, homogenizing the obtained spinal cord tissue, mashing the tissue in physiological saline, centrifuging for 10 min at 3000 r.f., and collecting supernatant. The required panels were taken out of the aluminum foil bags after equilibration for 20 minutes at room temperature, and the remaining panels were sealed with a valve bag and stored at 4 ℃.

Setting a standard substance hole and a sample hole, and adding 50 mu l of IgG solution standard substance with a determined known concentration into each standard substance hole; adding 10 mul of sample to be detected into the sample hole, and adding 40 mul of diluent into the sample hole; blank wells were not added. In addition to the blank wells, 100. mu.l of detection antibody labeled with horseradish peroxidase (HRP) was added to each of the standard wells and the sample wells, the reaction wells were sealed with a sealing plate film, and incubated in a water bath or incubator at 37 ℃ for 60 minutes. Discarding the liquid, patting the liquid on absorbent paper, filling the cleaning solution into each hole, standing for 1 minute, throwing off the cleaning solution, patting the liquid on the absorbent paper, and repeating the plate washing for 5 times. Substrate A (H) was added to each well₂O₂) 50. mu.l of each of (TMB/OPD) was incubated at 37 ℃ for 15 minutes in the absence of light. The OD of each well was measured at a wavelength of 450nm within 15 minutes by adding 50. mu.l of a stop solution to each well. Selecting a detection antibody sample which has positive binding with the induced NB-3 protein. As shown in fig. 6, fig. 6A shows that Elisa detects specific binding between each group of antibodies and the protein of contact 6 in spinal cord tissue, binds to the secondary antibody coupled to horseradish peroxidase through anti-mouse, and catalyzes a substrate color reaction, wherein rows a to C in fig. 6A represent that agtn 6-117, agtn 6-025 and agtn 6-027 are respectively expressed in sample stock solution, 1: the results of the color reaction at 5 dilutions and 1:10 dilutions, samples E-G in FIG. 6A represent the results of the color reaction for IgG control, and FIG. 6B is the results of absorbance of the color reaction by a microplate reader at 562nm, from which FIG. 6B it can be seen that the color reaction is stronger and thus the titer is relatively stronger for the last sample (aCNTN6-027) relative to the first two antibody detection samples (aCNTN6-117 and aCNTN 6-025).

(2) Western blot detection

Taking spinal cord tissues: a5 mm long tissue was cut from the eighth to tenth thoracic section of spinal cord tissue centered on the injured area to examine the protein expression level of the injured spinal cord and the binding ability of the corresponding antibody sample.

The tissue was mechanically triturated and dissolved in RIPA buffer (50mM Tris-HCl buffer, 150mM NaCl solution, 1mM EDTA sodium salt solution, 1% NP-40, 0.25% deoxycholate sodium solution, protease inhibitor cocktail and phosphatase inhibitor cocktail). After ultrasonic homogenization and heating, the resulting supernatant was subjected to protein gel electrophoresis, and then electrophoretically transferred to a PVDF membrane.

The PVDF membrane left with the protein marker was incubated with a primary antibody (NB-3 protein antibody from R & D, dilution ratio 1: 1000; beta-tubulin protein antibody from Sigma, dilution ratio 1: 1000; GAPDH protein antibody from Sigma, dilution ratio 1:1000) at 4 ℃ overnight in a refrigerator, the PVDF membrane left with the protein marker was incubated with a secondary antibody conjugated with horseradish peroxidase (GE healthcare) at room temperature for 1 hour, and finally an enhanced developer (Millipore) was applied to the PVDF membrane and placed in a gel imager from Bio-Rad to obtain the result of imaging the corresponding protein band. Selecting a detection antibody sample which has positive binding with the induced NB-3 protein. As shown in FIG. 7, the binding capacity of the last sample (aCNTN6-027) was relatively stronger than that of the first two antibody samples (aCNTN6-117 and aCNTN 6-025).

(3) Tissue culture technology for detecting suppression mediated by antibody blocking contact 6 homology interaction

Somatic motor cortex tissue culture: obtaining somatosensory motor cortex of an embryonic mouse from a mother mouse on the eighteenth day of pregnancy, separating centrum cells of the fifth and sixth layers, inoculating the centrum cells into a four-hole plate which is pre-paved with rat tail collagen mixed with a contact 6 protein, and culturing, wherein the used culture medium comprises the following components: cell culture medium (MEM), 5% Fetal Bovine Serum (FBS), 2% glucose, 1% glutamine solution, 1% penicillin, and streptomycin cocktail. After 60 hours of culture, the cells were fixed with 4% Paraformaldehyde (PFA) solution and immunofluorescent-stained with an antibody against β -tubulin III protein (Tuj1), and the results are shown in FIG. 8. It can be seen that the addition of NB-3-Fc protein blocked axonal growth of nerve tissue blocks (FIG. 8C) relative to the blank control group (FIG. 8B), and that the addition of the respective groups of blocking antibodies (aCNTN6-117, aCNTN6-025, aCNTN6-027, etc.) resulted in a somewhat successful block of NB-3-Fc inhibition (FIGS. 8D-F) by the corresponding final group of test antibodies (aCNTN6-027), and in vitro tissue culture promoted axonal regeneration.

(4) Implantable osmotic pressure pump technology for detecting inhibition mediated by antibody blocking contact 6 homology interaction

The experiment was carried out using wild type mice as the subjects (after which aptn 6-027 and control IgG antibody, etc. were pumped using an implantable osmotic pump), and the mice were kept in a pathogen free class (SPF) laboratory.

First, a mouse model was constructed using mice with a body weight of 20-25g and an age of 10 weeks after birth.

Before surgery, mice were intraperitoneally injected with 40 mg of sodium pentobarbital per kg of body weight and anesthetized. The spinal cord dorsal hemisection model was performed in the region of cervical spinal cord segment 4. After the injury model is applied, the ligament and the skin are sutured together. Mice were subjected to twice daily bladder compressions to promote urination after surgery and were injected intramuscularly with 10000 units of penicillin per kg of body weight.

After the mice are anesthetized by intraperitoneal injection, the intrathecal implant is as follows: the micro-catheter is placed about 2cm to the enlarged part of the cervical spine, and the micro-catheter is connected with a polyethylene tube and then is sewn layer by layer to fix the catheter. One million units of penicillin sodium per kilogram of body weight was used to prevent post-operative infection. After 3 days, when lidocaine is slowly administrated in the animal sheath without the catheter placing complication, paralysis of the upper limb of the animal shows that the catheter is in the subarachnoid space, and the experiment can be carried out. Before the implantable osmotic pressure pump is used, the implantable osmotic pressure pump is soaked in sterile normal saline, a polyethylene pipe is connected with the pump and is fully fixed, the implantable osmotic pressure pump is implanted into the subcutaneous back of a mouse, and then the implantable osmotic pressure pump is sutured well.

Antegrade marking of somatosensory motor cortex vertebral body neurons: a Bregma point on a mouse skull is taken as an origin, a parallel line passing through the Bregma point in parallel lines of two lugs is taken as a Y axis, a straight line which is perpendicular to the Y axis and passes through the Bregma point is taken as an X axis (the head end is the positive direction of the X axis), a coordinate system (the unit is mm) is established, four coordinate points (the injection depth is 0.5mm) of (1.0, 1.5), (0.5, 1.5) (-1.0,1.5) are selected as a single-side injection biotin-bonded dextran-glycosaminoglycan (BDA) (10% solution produced by Invitrogen company) on a somatosensory motor cortex, and each coordinate point is injected with 0.4 mu l. In order to detect the regeneration of the corticospinal tracts, biotin-bound glucosaminoglycamine (BDA) is injected into the somatosensory motor cortex of the mice at 12 weeks after surgery and survives for two weeks before the materials of the mice are sacrificed, so that the biotin-bound glucosaminoglycamine (BDA) can be marked to the corticospinal tracts which penetrate from the cortex to the lumbosacral segment of the spinal cord.

Fluorescence intensity analysis of biotin-bound dextran glycosaminoglycan (BDA) anterograde labeled axons: the results after immunofluorescent staining of spinal sagittal sections are shown in FIG. 9. In the figure, the A diagram represents the test group in which the in vivo pump continues the in vivo injection of the antibody (aCNTN6-027), and the B diagram represents the control group (IgG). The results in the figure show that injured corticospinal tract axons can promote axon regeneration to some extent in vivo conditions in the presence of antibody injection, relative to the control group.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

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Thr Val Lys Met Glu Cys Phe Ala Leu Gly Asn Pro Val Pro Thr

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Ile Thr Trp Met Lys Val Asn Gly Tyr Ile Pro Ser Lys Ser Arg

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Leu Arg Lys Ser Gln Ala Val Leu Glu Ile Pro Asn Leu Gln Leu

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Asp Asp Ala Gly Ile Tyr Glu Cys Thr Ala Glu Asn Ser Arg Gly

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Trp Val Gln Lys Leu Asn Asp Thr Gln Leu Asp Ser Gly Ser Pro

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Arg Trp Leu Lys Asn Gly Ala Pro Leu Leu Pro Gln Ser Arg Val

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Phe Glu Leu Asn Gln Val Lys Lys Ser Ile Ile Val Thr Lys Asp

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Ala Ile Ser Trp Arg Lys Gly Asp Lys Ala Val Arg Ala Asn Lys

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Arg Ile Ala Ile Leu Pro Asp Gly Ser Leu Arg Ile Leu Asn Ala

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Ser Lys Ala Asp Glu Gly Lys Tyr Ile Cys Gln Gly Val Asn Ile

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Glu Ser Ile Val Leu Asn Cys Lys Ala Ile His Asp Ala Ser Leu

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Asp Val Thr Phe Tyr Trp Thr Leu Lys Gly Gln Pro Ile Asp Phe

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Glu Lys Glu Gly Gly His Phe Glu Asn Ile Arg Ala Gln Ala Ser

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Ser Ala Asp Leu Met Ile Arg Asn Ile Leu Leu Met His Ala Gly

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Val Ile Val Glu Glu Ile Thr Glu Ser Thr Ala Thr Leu Ser Trp

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Ser Pro Ala Thr Asp Asn His Ser Pro Ile Ser Ser Tyr Asn Leu

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Gln Ala Arg Ser Pro Phe Ser Leu Gly Trp Gln Thr Val Lys Thr

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Val Pro Glu Val Ile Thr Gly Asp Met Glu Ser Ala Met Ala Val

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Asp Leu Asn Pro Trp Val Glu Tyr Glu Phe Arg Val Val Ala Thr

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Asn Pro Ile Gly Thr Gly Asp Pro Ser Ile Pro Ser Arg Met Ile

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Arg Thr Asn Glu Ala Val Pro Lys Thr Ala Pro Ser Asn Val Ser

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Gly Arg Ser Gly Arg Arg His Glu Leu Val Ile Ala Trp Glu Pro

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Val Ser Glu Glu Phe Gln Asn Gly Glu Gly Phe Gly Tyr Ile Val

800 805 810

Ala Phe Arg Pro Asn Gly Thr Arg Gly Trp Lys Glu Lys Met Val

815 820 825

Thr Ser Ser Glu Ala Ser Lys Phe Ile Tyr Arg Asp Glu Ser Val

830 835 840

Pro Pro Leu Thr Pro Phe Glu Val Lys Val Gly Val Tyr Asn Asn

845 850 855

Lys Gly Asp Gly Pro Phe Ser Gln Ile Val Val Ile Cys Ser Ala

860 865 870

Glu Gly Glu Pro Thr Ala Ala Pro Thr Asp Val Thr Ala Thr Ser

875 880 885

Val Ser Val Ser Glu Ile Phe Val Val Trp Lys His Val Lys Glu

890 895 900

Ser Leu Gly Arg Pro Gln Gly Phe Glu Ile Ser Tyr Trp Lys Asp

905 910 915

Thr Glu Pro Glu Asp Ser Val Glu Thr Val Arg Thr Arg Gly Asn

920 925 930

Glu Ser Phe Val Met Leu Thr Gly Leu Glu Gly Asn Thr Leu Tyr

935 940 945

His Leu Thr Val Arg Ala Tyr Asn Gly Ala Gly Tyr Gly Pro Pro

950 955 960

Ser Arg Glu Ala Ser Thr Thr Thr Lys Arg His Pro Pro Arg Glu

965 970 975

Pro Pro Gly Asn Leu Arg Trp Glu Gln Gln Gly Ser Gln Val Ser

980 985 990

Leu Gly Trp Glu Pro Val Arg Pro Leu Ala Asn Glu Ser Glu Val

995 1000 1005

Met Gly Tyr Lys Val Phe Tyr Arg Gln Glu Gly His Ser Glu Gly

1010 1015 1020

Gln Val Ile Glu Thr Gln Lys Pro Gln Ala Val Val Pro Leu Pro

1025 1030 1035

Glu Ala Gly Val Tyr Ile Ile Glu Val Arg Ala Tyr Ser Glu Gly

1040 1045 1050

Gly Asp Gly Thr Ala Ser Ser Gln Ile Arg Val Pro Ser Tyr Ser

1055 1060 1065

Gly Gly Lys Ile Thr Ser Ala Gln Ser Thr Leu His Ser Leu Ser

1070 1075 1080

Lys Trp Ser Ser Val Thr Leu Leu Leu Ala Leu Met Leu Pro Ser

1085 1090 1095

Ser Ser Trp

<210> 4

<211> 1028

<212> PRT

<213> amino acid sequence

<400> 4

Met Arg Leu Leu Trp Lys Leu Val Ile Leu Leu Pro Leu Ile Asn

5 10 15

Ser Cys Ala Gly Glu Gly Arg Phe Ser Arg Pro Ile Phe Ile Gln

20 25 30

Glu Pro Gln Asp Val Ile Phe Pro Leu Asp Leu Ser Arg Ser Glu

35 40 45

Ile Ile Leu Thr Cys Thr Ala Asn Gly Tyr Pro Ser Pro His Tyr

50 55 60

Arg Trp Lys Gln Asn Gly Thr Asp Ile Asp Phe Gly Met Thr Tyr

65 70 75

His Tyr Arg Leu Asp Gly Gly Ser Leu Ala Ile Ser Ser Pro Arg

80 85 90

Thr Asp Gln Asp Ile Gly Ile Tyr Gln Cys Leu Ala Thr Asn Pro

95 100 105

Val Gly Thr Ile Leu Ser Arg Lys Ala Lys Leu Gln Phe Ala Tyr

110 115 120

Ile Glu Asp Phe Glu Thr Lys Thr Arg Ser Thr Val Ser Val Arg

125 130 135

Glu Gly Gln Gly Val Val Leu Leu Cys Gly Pro Pro Pro His Phe

140 145 150

Gly Glu Leu Ser Tyr Ala Trp Thr Phe Asn Asp Ser Pro Leu Tyr

155 160 165

Val Gln Glu Asp Lys Arg Arg Phe Val Ser Gln Asp Thr Gly Asn

170 175 180

Leu Tyr Phe Ala Lys Val Glu Pro Ser Asp Val Gly Asn Tyr Thr

185 190 195

Cys Phe Val Thr Asn Lys Glu Ala His Arg Ser Val Gln Gly Pro

200 205 210

Pro Thr Pro Leu Val Leu Arg Thr Asp Gly Val Met Gly Glu Tyr

215 220 225

Glu Pro Lys Ile Glu Val Arg Phe Pro Glu Thr Ile Gln Ala Ala

230 235 240

Lys Asp Ser Ser Ile Lys Leu Glu Cys Phe Ala Leu Gly Asn Pro

245 250 255

Val Pro Asp Ile Ser Trp Lys Arg Leu Asp Gly Ser Pro Met Pro

260 265 270

Gly Lys Ile Lys Tyr Ser Lys Ser Gln Ala Ile Leu Glu Ile Pro

275 280 285

Lys Phe Gln Gln Glu Asp Glu Gly Phe Tyr Glu Cys Ile Ala Gly

290 295 300

Asn Leu Arg Gly Arg Asn Leu Ala Lys Gly Gln Leu Ile Phe Tyr

305 310 315

Ala Pro Pro Glu Trp Glu Gln Lys Ile Gln Asn Thr Tyr Leu Ser

320 325 330

Ile Tyr Asp Ser Leu Phe Trp Glu Cys Lys Ala Ser Gly Asn Pro

335 340 345

Asn Pro Ser Tyr Thr Trp Leu Lys Asn Gly Gln Arg Leu Asn Thr

350 355 360

Glu Glu Arg Ile Gln Ile Glu Asn Gly Thr Leu Ile Ile Thr Met

365 370 375

Leu Asn Ile Ser Asp Ser Gly Ile Tyr Gln Cys Ala Ala Glu Asn

380 385 390

Lys Tyr Gln Thr Ile Tyr Ala Asn Ala Glu Leu Arg Val Leu Ala

395 400 405

Ser Ala Pro Asp Phe Ser Lys Asn Pro Ile Lys Lys Ile Ser Val

410 415 420

Val Gln Val Gly Gly Asp Ile Ser Ile Glu Cys Lys Pro Asn Ala

425 430 435

Phe Pro Lys Ala Ser Ile Ser Trp Lys Arg Gly Thr Glu Asn Leu

440 445 450

Lys Gln Ser Lys Arg Val Leu Phe Leu Glu Asp Gly Ser Leu Lys

455 460 465

Ile Cys Asn Val Thr Arg Ala Asp Ala Gly Ser Tyr Thr Cys Val

470 475 480

Ala Thr Asn Gln Phe Gly Asn Gly Lys Ser Ser Gly Gly Leu Val

485 490 495

Val Lys Glu Arg Thr Ile Ile Thr Val Pro Pro Ser Lys Met Asp

500 505 510

Val Thr Val Gly Glu Ser Ile Val Leu Pro Cys Gln Val Ser His

515 520 525

Asp Pro Thr Met Glu Val Leu Phe Val Trp Tyr Phe Asn Gly Asp

530 535 540

Ile Ile Asp Leu Lys Lys Gly Val Ala His Phe Glu Arg Ile Gly

545 550 555

Gly Glu Ser Val Gly Asp Leu Met Ile Arg Asn Ile Gln Leu Gly

560 565 570

His Ser Gly Lys Tyr Leu Cys Thr Val Gln Thr Thr Leu Glu Arg

575 580 585

Leu Ser Ala Val Ala Asp Ile Ile Val Arg Gly Pro Pro Gly Pro

590 595 600

Pro Glu Asp Val Lys Val Glu His Ile Ser Ser Thr Thr Ser Gln

605 610 615

Leu Ser Trp Arg Pro Gly Pro Asp Asn Asn Ser Pro Ile Gln Ile

620 625 630

Phe Thr Ile Gln Thr Arg Thr Pro Phe Ser Val Gly Trp Gln Ala

635 640 645

Val Ala Thr Val Pro Glu Ile Leu Asn Gly Gln Thr Tyr Asn Ala

650 655 660

Thr Val Val Gly Leu Ser Pro Trp Val Glu Tyr Glu Phe Arg Val

665 670 675

Val Ala Gly Asn Asn Ile Gly Ile Gly Glu Pro Ser Lys Pro Ser

680 685 690

Glu Leu Leu Arg Thr Lys Ala Ser Val Pro Asn Val Ala Pro Gly

695 700 705

Asn Ile Asn Gly Gly Gly Gly Ser Arg Ser Glu Leu Val Ile Thr

710 715 720

Trp Glu Ala Ile Pro Glu Glu Leu Gln Asn Gly Glu Gly Phe Gly

725 730 735

Tyr Ile Val Met Phe Arg Pro Val Gly Thr Thr Ala Trp Met Lys

740 745 750

Glu Arg Val Ala Leu Val Glu Ser Ser Lys Phe Ile Tyr Arg Asn

755 760 765

Glu Ser Ile Met Pro Leu Ser Pro Phe Glu Val Lys Val Gly Val

770 775 780

Tyr Asn Asn Glu Gly Glu Gly Ser Leu Ser Thr Val Thr Ile Val

785 790 795

Tyr Ser Gly Glu Asp Glu Pro Gln Leu Ala Pro Arg Gly Thr Ser

800 805 810

Val Gln Ser Phe Ser Ala Ser Glu Met Glu Val Ser Trp Asn Ala

815 820 825

Ile Ala Trp Asn Arg Asn Thr Gly Arg Val Leu Gly Tyr Glu Val

830 835 840

Leu Tyr Trp Thr Asp Asn Ser Lys Glu Ser Met Ile Gly Lys Ile

845 850 855

Arg Val Ser Gly Asn Val Thr Thr Lys Asn Ile Thr Gly Leu Arg

860 865 870

Ala Asn Thr Ile Tyr Phe Ala Ser Val Arg Ala Tyr Asn Thr Ala

875 880 885

Gly Thr Gly Pro Ser Ser Leu Pro Val Asn Val Thr Thr Lys Lys

890 895 900

Ser Pro Pro Ser Gln Pro Pro Ala Asn Ile Ala Trp Lys Leu Ser

905 910 915

Asn Ser Lys Leu Cys Leu Asn Trp Glu His Val Lys Thr Met Glu

920 925 930

Asn Glu Ser Glu Val Leu Gly Tyr Lys Ile Leu Tyr Arg Gln Asn

935 940 945

Arg Gln Ser Lys Thr His Ile Leu Glu Thr Asn Asn Thr Ser Ala

950 955 960

Glu Leu Leu Val Pro Phe Glu Glu Asp Tyr Leu Ile Glu Ile Arg

965 970 975

Thr Val Ser Asp Gly Gly Asp Gly Ser Ser Ser Glu Glu Ile Arg

980 985 990

Ile Pro Lys Met Ser Ser Leu Ser Ser Thr Gly Val Gln Ile Ser

995 1000 1005

Lys Pro Ser Thr Gln Ser Leu Ser Met Val Gly Val Phe Tyr Cys

1010 1015 1020

Phe Ala Ile His Pro Leu Ser Arg

1025

<210> 5

<211> 197

<212> PRT

<213> amino acid sequence

<400> 5

Arg Lys Ala Lys Leu Gln Phe Ala Tyr Ile Glu Asp Phe Glu Thr

5 10 15

Lys Thr Arg Ser Thr Val Ser Val Arg Glu Gly Gln Gly Val Val

20 25 30

Leu Leu Cys Gly Pro Pro Pro His Phe Gly Glu Leu Ser Tyr Ala

35 40 45

Trp Thr Phe Asn Asp Ser Pro Leu Tyr Val Gln Glu Asp Lys Arg

50 55 60

Arg Phe Val Ser Gln Asp Thr Gly Asn Leu Tyr Phe Ala Lys Val

65 70 75

Glu Pro Ser Asp Val Gly Asn Tyr Thr Cys Phe Val Thr Asn Lys

80 85 90

Glu Ala His Arg Ser Val Gln Gly Pro Pro Thr Pro Leu Val Leu

95 100 105

Arg Thr Asp Gly Val Met Gly Glu Tyr Glu Pro Lys Ile Glu Val

110 115 120

Arg Phe Pro Glu Thr Ile Gln Ala Ala Lys Asp Ser Ser Ile Lys

125 130 135

Leu Glu Cys Phe Ala Leu Gly Asn Pro Val Pro Asp Ile Ser Trp

140 145 150

Lys Arg Leu Asp Gly Ser Pro Met Pro Gly Lys Ile Lys Tyr Ser

155 160 165

Lys Ser Gln Ala Ile Leu Glu Ile Pro Lys Phe Gln Gln Glu Asp

170 175 180

Glu Gly Phe Tyr Glu Cys Ile Ala Gly Asn Leu Arg Gly Arg Asn

185 190 195

Leu Ala

<210> 6

<211> 396

<212> PRT

<213> amino acid sequence

<400> 6

Pro Glu Asp Val Lys Val Glu His Ile Ser Ser Thr Thr Ser Gln

5 10 15

Leu Ser Trp Arg Pro Gly Pro Asp Asn Asn Ser Pro Ile Gln Ile

20 25 30

Phe Thr Ile Gln Thr Arg Thr Pro Phe Ser Val Gly Trp Gln Ala

35 40 45

Val Ala Thr Val Pro Glu Ile Leu Asn Gly Gln Thr Tyr Asn Ala

50 55 60

Thr Val Val Gly Leu Ser Pro Trp Val Glu Tyr Glu Phe Arg Val

65 70 75

Val Ala Gly Asn Asn Ile Gly Ile Gly Glu Pro Ser Lys Pro Ser

80 85 90

Glu Leu Leu Arg Thr Lys Ala Ser Val Pro Asn Val Ala Pro Gly

95 100 105

Asn Ile Asn Gly Gly Gly Gly Ser Arg Ser Glu Leu Val Ile Thr

110 115 120

Trp Glu Ala Ile Pro Glu Glu Leu Gln Asn Gly Glu Gly Phe Gly

125 130 135

Tyr Ile Val Met Phe Arg Pro Val Gly Thr Thr Ala Trp Met Lys

140 145 150

Glu Arg Val Ala Leu Val Glu Ser Ser Lys Phe Ile Tyr Arg Asn

155 160 165

Glu Ser Ile Met Pro Leu Ser Pro Phe Glu Val Lys Val Gly Val

170 175 180

Tyr Asn Asn Glu Gly Glu Gly Ser Leu Ser Thr Val Thr Ile Val

185 190 195

Tyr Ser Gly Glu Asp Glu Pro Gln Leu Ala Pro Arg Gly Thr Ser

200 205 210

Val Gln Ser Phe Ser Ala Ser Glu Met Glu Val Ser Trp Asn Ala

215 220 225

Ile Ala Trp Asn Arg Asn Thr Gly Arg Val Leu Gly Tyr Glu Val

230 235 240

Leu Tyr Trp Thr Asp Asn Ser Lys Glu Ser Met Ile Gly Lys Ile

245 250 255

Arg Val Ser Gly Asn Val Thr Thr Lys Asn Ile Thr Gly Leu Arg

260 265 270

Ala Asn Thr Ile Tyr Phe Ala Ser Val Arg Ala Tyr Asn Thr Ala

275 280 285

Gly Thr Gly Pro Ser Ser Leu Pro Val Asn Val Thr Thr Lys Lys

290 295 300

Ser Pro Pro Ser Gln Pro Pro Ala Asn Ile Ala Trp Lys Leu Ser

305 310 315

Asn Ser Lys Leu Cys Leu Asn Trp Glu His Val Lys Thr Met Glu

320 325 330

Asn Glu Ser Glu Val Leu Gly Tyr Lys Ile Leu Tyr Arg Gln Asn

335 340 345

Arg Gln Ser Lys Thr His Ile Leu Glu Thr Asn Asn Thr Ser Ala

350 355 360

Glu Leu Leu Val Pro Phe Glu Glu Asp Tyr Leu Ile Glu Ile Arg

365 370 375

Thr Val Ser Asp Gly Gly Asp Gly Ser Ser Ser Glu Glu Ile Arg

380 385 390

Ile Pro Lys Met Ser Ser

395

Claims (5)

1. An antigen of the neural recognition molecule contitin 6, characterized in that: the amino acid sequence of the antigen is shown as SEQ ID No. 1.

2. An antibody of the neural recognition molecule contitin 6, characterized in that: the antibody is prepared from the antigen of claim 1.

3. The antibody of claim 2, wherein: the antibody is a monoclonal antibody.

4. The antibody of claim 2 or 3, wherein the antibody is prepared by a method comprising the steps of:

(1) immunizing a mouse by using a contictin 6 antigen, and stimulating B lymphocytes of the mouse to clone and differentiate into sensitized B lymphocytes; the amino acid sequence of the antigen is shown as SEQ ID No. 1;

(2) extracting splenocytes of the mice, and mixing myeloma cells with the splenocytes to form hybridoma cells;

(3) culturing the hybridoma cells in HAT culture medium by adopting a limiting dilution method, and screening out positive hybridoma cells;

(4) and (3) proliferating the positive hybridoma cells in a mouse to obtain the antibody.

5. Use of an antibody according to claim 2 or 3 for the manufacture of a medicament for the treatment of spinal cord injury.