CN113527479A

CN113527479A - Antibody or antibody fragment specifically binding to voltage-gated sodium ion channel alpha subunit Nav1.7

Info

Publication number: CN113527479A
Application number: CN202010306517.9A
Authority: CN
Inventors: 杨代常
Original assignee: Eason Wuhan Biomedical Co Ltd
Current assignee: Eason Wuhan Biomedical Co Ltd
Priority date: 2020-04-17
Filing date: 2020-04-17
Publication date: 2021-10-22
Anticipated expiration: 2040-04-17
Also published as: CN113527479B

Abstract

The invention provides an antibody or an antibody fragment targeting a cell membrane voltage-gated sodium ion channel alpha subunit Nav1.7, wherein a specific binding target is an ion-conducting Pore Module (PM) of an S3 Domain of an IV Domain (Domain IV) of the voltage-gated sodium ion channel alpha subunit. The antibody or the antibody fragment can inactivate the ion conduction pore module, so that sodium ions can not normally enter nerve cells, thereby achieving the effects of treating and relieving pain.

Description

Antibody or antibody fragment specifically binding to voltage-gated sodium ion channel alpha subunit Nav1.7

Technical Field

The invention belongs to the field of biological medicine, and relates to an antibody and/or an antibody fragment which takes an ion conduction pore module of Nav1.7 as a target spot and specifically recognizes the target spot (polypeptide).

Background

Pain originates in nociceptors of the peripheral nervous system, and peripheral nervous tissue, as a kind of free nerve endings, is widely distributed in the skin, muscle, joint and visceral tissues throughout the body, and can convert a sensed thermal, mechanical or chemical stimulus into an action potential, which is transmitted to its somatic part located in the Dorsal Root Ganglion (DRG) through nerve fibers, and finally to the higher nerve center, thereby inducing pain sensation. The generation and conduction of action potentials in neurons, in turn, depend on voltage-gated sodium channels (VGSCs) located on the cell membrane. When the cell membrane depolarizes, the sodium ion channel is activated and opened to cause the internal flow of sodium ions, which further depolarizes the cell membrane, resulting in the generation of action potential and pain due to abnormal action potential. Thus, inhibition of aberrant sodium ion channel activity contributes to the treatment or alleviation of pain.

Voltage-gated sodium ion channels can be classified into 9 subtypes, and at present, 9 subtypes of voltage-gated sodium ion channels have been identified in mammals, because the amino acid sequence similarity is greater than 50%. Therefore, they are considered to be derived from the same family and named Nav1(Nav1.1 to Nav 1.9). Voltage-gated sodium ion channels are widely present in cell membranes of cells such as neurons and skeletal muscle cells, and are a type of transmembrane glycoprotein complex composed of an α subunit having 2 functional domains (domains), i.e., ion-conducting pore domains (ion-conducting domains) and voltage-sensing domains (VSDs), and a plurality of auxiliary β subunits, wherein the α subunit has pores formed by 4 repetitive domains (DI-DIV), each of which contains 6 transmembrane helical segments (S1-S6). S1-S4 contain voltage sensing domains VSD, and S5-S6 constitute tetramer conformation pore-forming domains. In the VSD domain, S4 contains VSD and S4 is rich in arginine with gated charge, can sense membrane potential changes, which together with the C-terminus of S3 form a voltage-sensor paddle (voltage-sensor paddle), whose motion reflects changes in membrane potential and couples to the opening, closing and inactivation of the aperture. The paddle movement is induced to open and lock the channel due to this voltage. Therefore, this functional domain is an important target for drug action, and can regulate the switching of channels through protein interaction.

Recent studies have shown that the subtypes of Nav1 associated with pain are primarily Nav1.7, Nav1.8, and Nav 1.9. Among them, Nav1.7 is mainly responsible for pain of one of the important members. Nav1.7 is TTX-S type, and the coding gene is SCN 9A, mainly distributed in peripheral primary sensory neurons and sympathetic ganglion neurons, and participates in human pain signal pathways. Recently in the human pain-free patients occurred in Nav1.7 genetic mutation, produced pain-free symptoms; further studies have shown that this gene is one of the sodium ion channels primarily responsible for pain.

Small chemical molecules (e.g., carbamazepine, lidocaine, mexiletine, etc.) are widely used clinically as voltage-gated sodium ion channel inhibitors for the treatment of pain, but they lack sufficient selectivity for the subtype of voltage-gated sodium ion channel, thus causing the disadvantages of cardiotoxicity and central nervous side effects. Recently, some small molecule blockers (blocker) aiming at Nav1.7 enter clinical research, and because the subtype homology of sodium ion channels is high, the selectivity of the small molecule blockers is poor, and the side effects are difficult to overcome. However, the blocking agent of the macromolecule has high specificity, good stability and small side effect, but the voltage-gated sodium ion channel antigen for generating the antibody is difficult to prepare, so the research is very difficult.

Disclosure of Invention

An object of the present invention is to provide an antibody or an antibody fragment specifically binding to an α subunit nav1.7 of a voltage-gated sodium ion channel, wherein the specific binding takes an ion-conduction Pore Module (PM) of a DIVS3 domain in nav1.7 as a target, a polypeptide is designed by using the target of the region as an antigen to obtain a monoclonal antibody, and the normal state of VGSCs ion channels can be interfered by the binding of a specific antibody and the target thereof, so as to inhibit pain.

The second purpose of the invention is to provide a pharmaceutical composition containing the antibody or the antibody fragment of the ion conduction pore module which specifically binds to DIVS3 domain of voltage-gated sodium ion channel alpha subunit Nav1.7.

The third purpose of the invention is to provide the antibody or the antibody fragment thereof of the ion conduction pore module specifically binding to DIVS3 domain of the voltage-gated sodium ion channel alpha subunit Nav1.7 or the application of the pharmaceutical composition.

The invention also provides a nucleotide for coding the antibody or the antibody fragment, an expression vector containing the nucleotide and a preparation method of the antibody or the antibody fragment.

According to one aspect of the invention, the antibody or antibody fragment thereof of the invention specifically binds to the ion conduction pore module of the DIVS3 domain of voltage-gated sodium ion channel alpha subunit Nav1.7, and the specifically binding target is the ion conduction pore module of the DIV/S3 domain of voltage-gated sodium ion channel alpha subunit. More preferably, the antigen to which it binds has the amino acid sequence: DSVNVDKQPKYEYS (SEQ ID NO.9)

According to the crystal structure of Nav1.7, a polypeptide with good hydrophilicity and high antigenicity is selected as an antigen by screening a polypeptide with a suitable target area through an ion conduction pore module of a DIV/S3 structural domain of a voltage sensor valve of Nav1.7, and the amino acid sequence of the polypeptide is DSVNVDKQPKYEYS (SEQ ID NO.9) through the analysis of hydrophilicity and antigenicity.

Chemically synthesizing the polypeptide, numbering the synthesized polypeptide as C9797BL020-7(SEQ ID NO.9), coupling the polypeptide to a carrier protein KLH, immunizing a BALB/C mouse, inoculating an immune stimulator for multiple times to generate immune response so as to generate a polyclonal antibody, and performing blood sampling test, ELISA detection and evaluation.

Based on antigen-antibody reaction, evaluating the titer of polyclonal antibody generated by immune animals by ELISA, finally determining three animals #4061, #4062 and #4063 which meet the requirements according to the titer of the antibody of the immune animals and the specificity of human nervous tissue for cell fusion, taking spleen cells of the three animals and mouse myeloma cells (SP2/0) for cell electrofusion, performing cell culture after fusion, screening positive cell strains on a screening culture medium, screening hybridoma cell strains by using polypeptide C9797BL020-7 as an antigen, and selecting the positive cell strains for subcloning according to the titer of the antibody and the specificity of the human nervous tissue according to the result of ELISA detection. And performing ELISA detection and nerve tissue specificity detection on the obtained subclones, selecting the subclones with good nerve tissue specificity, and performing cell cryopreservation.

Extracting total RNA of cell strain, synthesizing cDNA, establishing cDNA library, and sequencing variable region. Amplifying polynucleotide sequences encoding the variable regions of the antibody, either by integrating DNA sequences encoding VH and VL (which may also be manipulated with RNA sequences encoding the variable regions) into the same vector, or by integrating them separately into a vector, and transfecting a suitable host cell with said vector; it was then subjected to sequencing analysis. The sequencing result shows that the DNA sequence of VH is shown as SEQ ID NO. 10, and the DNA sequence of VL is shown as SEQ ID NO. 11.

Constructing a genetic engineering antibody, introducing the DNA sequences coding for VH and VL (or coding for CDR in VH and coding for CDR in VL) into a suitable host according to different requirements, expressing the antibody, and verifying the antibody effect.

Detecting the immunogenicity of the monoclonal antibody, performing prokaryotic expression on a target partial sequence, extracting total protein from prokaryotic expression bacteria, performing primary purification to obtain an antigen fragment, and analyzing the binding specificity of the antibody by using a Western Blotting method, wherein the antibody can specifically recognize the target protein sequence of Nav1.7, as shown in figure 4. The mouse is induced to be molded by acute inflammatory pain by 5 percent formalin, and a proper amount of antibody is injected through tail vein to detect the analgesic effect of the antibody on the pain model mouse. The results are shown in fig. 5, and the injection of 10mg/kg antibody has significant analgesic effect compared with the control.

According to another aspect of the invention, the antibody or antibody fragment thereof of the invention specifically binding to the α subunit nav1.7 of a voltage-gated sodium ion channel comprises:

heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of the HCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.2, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3; and

the light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO.4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6.

According to the invention, the antibody is a monoclonal antibody or a polyclonal antibody. Preferably, the antibody is a monoclonal antibody.

According to the present invention, the antibody is a murine antibody, a chimeric antibody, a humanized antibody or the like. Preferably, the antibody is a humanized antibody.

According to the present invention, the antibody fragment includes Fab, F (ab') 2, dsFv, scFv, diabody, minibody, diabody, multispecific antibody, chimeric antibody, CDR-grafted antibody and the like.

According to a preferred embodiment of the present invention, the antibody or antibody fragment specifically binding to α subunit Nav1.7 of voltage-gated sodium ion channel comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8. It will be appreciated by those skilled in the art that the antibody or antibody fragment of the present invention also includes derivative sequences that are 80%, 80-85%, 85-90%, 90-95% or 95-99% homologous to the sequences shown in SEQ ID NO 7/8 of the heavy/light chain variable region described above.

According to a preferred embodiment of the invention, the antibody or antibody fragment that specifically binds to the α subunit Nav1.7 of a voltage-gated sodium ion channel comprises a heavy chain constant region and a light chain constant region, which may be of a species origin selected from the group consisting of: the antibody constant region comprises a human antibody constant region, a bovine antibody constant region, a ovine antibody constant region, a canine antibody constant region, a porcine antibody constant region, a feline antibody constant region, an equine antibody constant region and an donkey antibody constant region. Preferably, the heavy chain constant region is selected from the group consisting of an IgG1, IgG2, IgG3 and IgG4 heavy chain constant region and the light chain constant region is selected from the group consisting of a kappa or lambda light chain constant region. Preferably, the heavy chain constant region is an IgG4 heavy chain constant region and the light chain constant region is a kappa light chain constant region.

According to a preferred embodiment of the invention, the antibody or antibody fragment specifically binding to the α subunit nav1.7 of a voltage-gated sodium ion channel comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain being as set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 15.

According to a further aspect of the invention, there is provided a nucleotide sequence encoding an antibody or antibody fragment according to the invention that specifically binds to the α subunit nav1.7 of a voltage-gated sodium ion channel.

According to a preferred embodiment of the invention, the nucleotide sequence comprises: as shown in SEQ ID NO:10, as shown in SEQ ID NO:11, and a nucleotide sequence encoding a light chain variable region.

According to a preferred embodiment of the invention, the nucleotide sequence comprises: as shown in SEQ ID NO: 16, as shown in SEQ ID NO: 17 encoding a light chain.

The invention also provides an expression vector, which contains the nucleotide sequence as described in any one of the above.

The invention also provides a host cell containing the expression vector.

The invention also provides a method for preparing the antibody or the antibody fragment specifically binding to the alpha subunit Nav1.7 of the voltage-gated sodium ion channel, which comprises the following steps:

(a) culturing a host cell as described above under expression conditions such that said antibody or antibody fragment that specifically binds to the α subunit nav1.7 of the voltage-gated sodium ion channel is expressed;

(b) isolating and purifying the antibody or antibody fragment of (a) that specifically binds to voltage-gated sodium ion channel alpha subunit Nav1.7.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the antibody or antibody fragment specifically binding to the α -subunit of voltage-gated sodium ion channel, nav1.7, as an active ingredient, and a pharmaceutically acceptable carrier, which has analgesic and pain threshold increasing effects and is capable of treating pain, itch and cough.

According to a further aspect of the invention, there is provided a use of the antibody or antibody fragment specifically binding to the α -subunit Nav1.7 of a voltage-gated sodium ion channel or the pharmaceutical composition of the invention in the preparation of a medicament for the treatment of a pain-related disorder.

Has the advantages that: the biomacromolecule, an antibody, is adopted to specifically bind to the target of a voltage sensor of a Nav1.7 voltage-gated sodium ion channel to inactivate an ion conduction pore module of a DIVS3 structural domain, so that normal sodium ions cannot enter nerve cells, and the effects of treating and relieving pain are achieved.

Drawings

FIG. 1: the structure diagram and the target point schematic diagram of a sodium ion channel Nav 1.7;

FIG. 2: immunohistochemical analysis of human neural tissue specificity of the sera of the immunized animals;

A、4061；B、4062；C、4063；D、4064；E、4065

FIG. 3: western blotting immunogenicity analysis of the monoclonal antibody;

FIG. 4: SPR measurement Nav1.7 target monoclonal antibody 5C12D2C8 affinity binding curve;

FIG. 5: nav1.7 targeting monoclonal antibody 5C12D2C8 in human peripheral nerve cells localization;

FIG. 6: analgesic effect of 5C12D2C8 antibody on acute inflammatory pain induced by 5% formalin in wild type mice.

Detailed Description

The following detailed description of the preferred embodiments of the invention, while not limiting the invention, illustrates the invention.

Material sources are as follows:

the materials and reagents used below were all purchased commercially unless otherwise specified.

Example 1 antigen Synthesis

Based on Nav1.7 amino acid sequence (GenBank No. NP-002968) and crystal structure functional region (FIG. 1), hydrophilic and antigenic analysis is carried out, DSVNVDKQPKYEYS sequence is screened out, the hydrophilic and antigenic meet the requirement of antigen, CDSVNVDKQPKYEYS (SEQ ID NO.9) polypeptide is artificially synthesized by adopting a full-automatic synthesizer.

The method comprises the following specific steps:

(1) linking the first AA-COOH to Cl-Resin with DIEA, and blocking the unreacted functional groups on the Resin with MeOH;

(2) washing with DMF;

(3) removal of-NH from the first AA with Pip₂Protecting group Fmoc of (2) to-NH₂Exposing;

(4) washing with DMF;

(5) activation of the second AA-COOH by DIC + HOBTThen condensed to-NH in the first AA₂To form an amide bond;

(6) washing with DMF;

(7) removal of-NH from the second AA with Pip₂Protecting group Fmoc of (2) to-NH₂Exposing;

(8) washing with DMF;

(9) … repeat 5-8 until the last AA-NH₂Exposing;

(10) and (3) cleaving the polypeptide from the resin and cutting off the side chain protecting groups of all amino acids by using a cleavage reagent which is: trifluoroacetic acid, ethanedithiol, phenol, thioanisole and water;

(11) adding the cutting fluid into ether to precipitate the polypeptide, and centrifuging to obtain crude polypeptide (C9797BL 020-7);

(12) polypeptide HPLC C18 preparative/analytical column was purified, and the purified polypeptide was obtained under the number C9797BL020-7 and used for immunizing animals.

Note: to facilitate conjugation of the polypeptide, a cysteine may be additionally added to the end of the polypeptide.

EXAMPLE 2 preparation of monoclonal cell lines

2.1 animal immunization

Freund's complete adjuvant Sigma, F5881 and Freund's incomplete adjuvant (Sigma, F5506) were prepared. The polypeptide is coupled to a carrier protein KLH by utilizing the terminal-SH of the polypeptide C9797BL020-7 to be used as immunogen.

5 female BALB/c (animal numbers: #4061, #4062, #4063, #4064, #4065) aged for 8 weeks were selected and subjected to 3 intraperitoneal immunizations, which stimulated the body to generate immune response and thus produced antibodies. Initial immune: 50 mu g/mouse, and performing secondary immunization after three weeks, wherein the dose is 50 mu g/mouse; the third immunization was carried out 2 weeks after the second immunization at a dose of 50. mu.g/mouse, and blood was collected 1 week after the third immunization for antibody detection.

2.2 animal serum ELISA detection

2.2.1 instruments and devices:

washing the plate machine: beijing Naohua ZDMX

An enzyme-labeling instrument: thermo MultiskanAscent

2.2.2 reagents used:

the coating antigen is polypeptide C9797BL 020-7; coating solution is 1 PBS (pH7.4); washing buffer solution: 1 × PBS (ph7.4), 0.05% PBS; the primary antibody is 3-immune antiserum; enzyme-labeled secondary antibody: Peroxidase-Affinipure Goat Anti-Mouse IgG, Fc gamma Fragment Specific (min X Hu, Bov, HrsSrProt); TMB color development liquid; stopping liquid: 1M hydrochloric acid.

The specific method comprises the following steps:

(1) coating: the antigen was diluted to 1. mu.g/ml with the coating solution, mixed well and added to the plate in 100. mu.l per well, covered with a cover plate membrane and left overnight at 4 ℃.

(2) And (3) sealing: taking out the plate, removing the coating solution, adding the confining solution, covering with a cover plate film, and keeping the temperature constant temperature box at 37 ℃ for 0.5 h.

(3) Adding a primary antibody: the 3-immune antiserum is diluted by first hole 1/1000, then diluted by multiple ratio for 9 gradients, covered with a cover plate film, and kept in a constant temperature box at 37 ℃ for 1 h.

(4) Adding a secondary antibody: taking out the enzyme-linked plate, discarding the internal solution, adding diluted enzyme-linked secondary antibody with the concentration of 0.033 mu g/ml, covering with a cover plate film, and keeping in a constant temperature box at 37 ℃ for half an hour.

(5) Color development: taking out the enzyme label plate, discarding the inner solution, adding color development solution, and developing at 25 deg.C for 13 min.

(6) And (4) stopping the reaction by adding a stopping solution.

(7) Immediately after the stop solution is added, reading at 450nm on a microplate reader, determining the maximum dilution corresponding to the well with OD value greater than 2.1 times of the set negative control OD value as the titer of the sample, wherein the detection result is shown in Table 1, NC is the negative control of the immune serum, and the initial dilution multiple is 1:1,000. The antiserum after three times of immunization is detected, and the antiserum titer of animal numbers #4061, #4062 and #4063 is 1:512,000; the S/B value was highest, and the antiserum titers of the remaining 2 animals (#4064, #4065) were 1:256,000, but the S/B values were only 2.7 or less; meanwhile, the serum is subjected to the histochemical analysis of human nerve cells, and the serum of three mice can have better histochemical signals (figure 2). Therefore, 3 animals were selected for cell fusion.

TABLE 1 serum ELISA test results after the third immunization

An animal number.	#4061	#4062	#4063	#4064	#4065
						Blank control	0.063	0.063	0.063	0.063	0.063
1:1,000	2.707	2.783	2.674	2.632	2.541
						1:2,000	2.606	2.58	2.532	2.438	2.405
1:4,000	2.571	2.28	2.405	2.345	2.319
						1:8,000	2.44	2.249	2.213	1.897	1.792
1:16,000	2.206	2.01	1.934	1.624	1.435
						1:32,000	1.888	1.62	1.566	1.246	1.003
1:64,000	1.479	1.227	1.199	0.843	0.677
						1:128,000	1.087	0.833	0.826	0.529	0.403
1:256,000	0.733	0.512	0.528	0.306	0.24
						1:512,000	0.435	0.313	0.306	0.174	0.165
Titer of the product	1:512,000	1:512,000	1:512,000	1:512,000	1:512,000
						S/B	6.905	4.968	4.857	2.762	2.619

2.3. Cytological specificity confirmation

To confirm whether these sera from stimulated immunization are specific for neural tissue, immunohistochemical analysis of human neural tissue was performed on 5 animal sera. The specific experimental method is as follows:

2.3.1 tissue dehydration treatment:

taking human nervous tissue slices for dehydration treatment, wherein the dehydration treatment adopts come card ASP300S, and the specific flow is as follows: respectively dehydrating for 30 minutes by adopting 70 percent, 85 percent and 90 percent of absolute ethyl alcohol; dehydrating with anhydrous ethanol for 2 times, each for 60 min; then treating for 30 minutes by using a clearing agent, and then treating for 2 times by using the clearing agent, wherein each time lasts for 60 minutes; and then processing with paraffin for 3 times, respectively 60 min, 120 min and 180 min, embedding with Leica EG1150 embedding machine to obtain wax block, and slicing to obtain slices with thickness of 4 μm.

2.3.2 in situ hybridization:

baking human nervous tissue slices at 85 deg.C for 20 min; the dewaxing agent is treated for 3 times, and each time lasts for 1 minute; dewaxing with anhydrous alcohol for 3 times, each time for 1 minute; washing with water for 3 times, each for 1 min; performing heat restoration by using ER2(PH is 9 buffer solution) for 20 minutes, cooling for 12 minutes, and washing with water for 3 times, wherein each time is 1 minute; then sealing for 30 minutes; washing with water for 3 times, each for 1 min; adding the cell strain supernatant, incubating for 30 minutes, washing with water for 3 times, each time for 1 minute; adopting an enhancer to incubate for 8 minutes, washing for 3 times, washing for 2 minutes each time, and adding a secondary antibody to incubate for 8 minutes; washing with water for 3 times, each for 2 minutes; DAB color development is carried out for 8 minutes; washing with water for 3 times, each for 1 min, and staining with hematoxylin for 10 min; washing with water for 3 times, each for 1 min, dehydrating with alcohol, and air drying. Observations were made under an olympus optical microscope.

The serum of 5 animals was positive to the human nervous tissue by observation with an optical microscope, indicating that the immunized animals produced antibodies specific to the nervous tissue (see FIG. 2).

2.4 cell fusion

Three animals #4061, #4062 and #4063 were selected for immunization based on ELISA assay results in combination with human neural tissue specificity assay results, spleen cells and tumor cells of the two animals were taken three days later for fusion, and mouse myeloma cells (SP2/0) and spleen cells were mixed in a ratio of 1: 3 ratio, performing cell fusion by electrofusion, and spreading the fused cells into 15 feeding blocks with HAT mediumPlacing CO into the cell culture plate₂Culturing in an incubator.

2.5 hybridoma cell strain screening:

after the fused cells are cultured for 7-10d, the culture solution is replaced by the whole plate, and ELISA is adopted for detection after the culture solution is replaced for 4 h. The ELISA specific materials and procedures were the same as the animal serum ELISA assays described in 2.2.

Each animal was plated 10, 40 total 96-well plates, 3840 total wells, and fusion animal serum 1:1000 dilutions were set as negative control and added to Blank medium, and when the OD value of the Blank was used for detection, the antibody titer was highest and S/B (Signal/Blank) > 2.1, positive clones were selected and then screened to obtain 135 candidate clones for the next round of subcloning.

2.5.1 subcloning:

the clones of 135 single-well cells selected for the first time were tested for the second time (the test method is the same as above), and 14 candidate clones were selected from the 135 candidate clones based on the antibody titer and the S/B ratio, and the test results of the 14 clones are shown in Table 2. These 14 clones will be used for further screening.

2.6. Affinity ranking:

in order to screen monoclonal antibody cell strains with relatively high affinity, 14 monoclonal antibody cell strains were subjected to affinity ranking. As shown in Table 3, the 14 monoclonal antibodies only have the three clones of 5C12D2C8,18F9G6D5 and 55F8C3B2 with higher reliability of fitting curves, wherein the affinity of 5C12D2C8 is the highest, and KD (M) reaches 4.55x10^-9Rmax (RU) reached only 291.1 (Table 3), and the cell line of this clone was used as a candidate clone.

TABLE 2 second round subclone screening (ELISA)

TABLE 3 affinity ranking results for antigen-antibody binding

Example 3 detection of antibody immunogenicity

3.1 antigen preparation

3.1.1: vector construction and crude protein preparation

Synthesizing 80 amino acid polypeptides containing an ion conduction pore module target of a DIVS3 structural domain of Nav1.7, constructing a prokaryotic expression His fusion protein expression vector, inoculating the prokaryotic expression His fusion protein expression vector into 2000mL LB liquid medium (kana resistance), performing shake culture at 37 ℃ overnight, and reducing the culture temperature to 30 ℃ when the OD600 is about 0.6; adding IPTG inducer to the final concentration of 0.1mM, and continuing shaking culture at 30 ℃ for 8 h; centrifuging for 3min, collecting thallus, suspending in 50mL precooled NTA-0 buffer solution, carrying out ice bath for 30min, carrying out ultrasonic cell disruption, centrifuging for 50min at 4 ℃, and collecting precipitate (inclusion body); the pellet was resuspended in 50mL NTA-0 buffer and DTT was added to a final concentration of 1 mM; ultrasonically promoting the dissolution of hybrid protein, centrifuging at 4 ℃ for 10min, and removing supernatant to obtain crude protein preparation solution;

3.1.2 protein denaturation and renaturation

Diluting a protein solution by using 2 times of volume of 3M guanidine hydrochloride for denaturation, taking the protein solution, putting the protein solution into a dialysis bag, concentrating the volume to 50-100 mL by using PEG20000, dialyzing the protein solution overnight by using PBS buffer solution at 4 ℃ for renaturation, and then concentrating the protein.

3.1.2 protein purification

Preparing Ni-NTA column according to manufacturer's instruction, and purifying protein according to the instruction

3.2 Western blot (WesternBlotting)

(1) Preparing separation gel according to a 12% separation gel formula;

(2) taking the corresponding antigen and the loading buffer 4: 1, mixing, and denaturing at 95 ℃ for 5 min;

(3) the amount of the antigen was staggered in lanes according to 4. mu.l Marker, 50. mu.l antigen;

(4) the voltage of 90V is used for running to the tail end of the concentrated gel, and the voltage is switched to 120V for running to the tail end 2/3 of the separation gel;

(5) taking out the gel, removing the concentrated gel, soaking the cut PVDF membrane in methanol for 2min, and then putting the gel, the PVDF membrane, the cut 6 pieces of filter paper and the support pad into a membrane transfer buffer solution for balancing for 10 min;

(6) assembling the cathode → the blackboard → the support pad → 3 pieces of filter paper → glue → the film → 3 pieces of filter paper → the support pad → the whiteboard → the anode in the sequence, and removing bubbles by using a glass rod when a layer is not assembled;

(7) fully adding a membrane-rotating buffer solution into the membrane-rotating tank, rotating the membrane for 30min at a voltage of 100V, and putting the membrane-rotating tank into ice water;

(8) taking out the film, dyeing with ponceau dye solution for 5min, slightly washing with water to see whether film transfer is successful, and washing ponceau with washing solution after strips are formed;

(9) soaking the membrane with sealing solution, sealing on shaking table for 1 hr, and washing with washing solution for 5min for 3 times;

(10) cutting the membrane into 5 strips, taking 5 different hybridoma supernatants, diluting the supernatants with PBS according to the proportion of 1 to 10 to be used as a primary antibody, and incubating the supernatants at 4 ℃ overnight;

(11) washing with washing solution for 5min for 3 times the next day, adding washing solution according to the ratio of 1: HRP-goat anti-mouse IgG diluted in 5000 proportion is incubated for 1h at room temperature;

(12) washing with washing solution for three times, dripping prepared DAB working solution onto the membrane after 10min each time, and observing the result after shading and developing for 8 min.

Western Blot results

The monoclonal antibody 5C12D2C8 of Nav1.7 was subjected to WesternBlotting hybridization with overlaid target polypeptide antigens, the results of which are shown in FIG. 3. As can be seen from the results of fig. 3: the 5C12D2C8 can recognize target polypeptide antigen signals, has antigen-antibody immune reaction, and proves that the 5C12D2C8 can recognize targets and has better specificity.

Example 4 antibody sequencing

To determine the monoclonal antibody sequence, monoclonal 5C12D2C8 was sequenced. Total RNA was isolated from hybridoma cells according to the technical manual of TRIzol reagent. Total RNA is then reverse transcribed into cDNA using either isotype specific antisense primers or universal primers, following the PrimeScript TM first strand cDNA synthesis kit technical manual. Antibody fragments for VH and VL were amplified according to the Standard Operating Procedure (SOP) method of GenScript Rapid Amplification of CDNA Ends (RACE). The amplified antibody fragments were cloned into standard cloning vectors, respectively. Colony PCR was performed to screen clones with the correct size insert. At least 5 colonies with the correct size insert were sequenced. The sequences of the different clones were aligned and the consensus sequences of these clones were determined.

The DNA sequence of VH is thus determined and is shown in SEQ ID NO 10; the DNA sequence of VL was determined as shown in SEQ ID NO 11.

The DNA sequence of the heavy chain variable region (VH) of monoclonal antibody 5C12D2C8 is shown in SEQ ID NO: 10; the DNA sequence of the light chain variable region (VL) is shown in SEQ ID NO: 11. VH and VL contain three Complementarity Determining Regions (CDRs), respectively, in the following positional relationships: wherein the leader sequence (signal peptide) is underlined in dotted lines, the CDR sequences are underlined in solid lines, and the Framework Regions (FR) are in bold.

Heavy chain:

-FR1-CDR1-FR2-CDR2-FR3-CDR3FR 4-constant region-termination codon heavy chain variable region (VH):

GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCGGCGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAATGGTGGTGGTGAGACCTACTATGAGGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTACAAATGAACAGTCTGAAGTCTGAGGACACAGCCAT

GTATTACTGTGCTAGGGAGGAGTACGAGGGTACGTGGTACTGAGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO:10) heavy chain constant region:

GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCC

AAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTAC

ACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTG

CCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTG

CATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACC

ATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGT

TCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAA

CAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTC

AAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCA

GACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAG

TCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAG

CCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCA

AGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGG

CCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA

light chain:

-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4-constant region-stop codon light chain variable region (VL):

GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCACATCTAGTCAGAGCGGAGTACATAGTAATGGAAACACCGAGTTAGAATGGTACCTGCAGAAACCAGGCCAGTCT

CCAAAGCTCCTGATCTACAAAGTTTCCAACCGAACATCTGGGGTCCCAGACAGGTTCAGTGGCAGTG

GATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCGGCCAAGGTTCACATGTTGGCCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ IDNO:11)

light chain constant region:

CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACG

ACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGC

ACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGA

CATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG

the amino acid sequence is deduced according to the DNA sequence, the VH amino acid sequence is shown as SEQ ID NO.7, and the VL amino acid sequence is shown as SEQ ID NO. 8.

Heavy chain:

-FR1-CDR1-FR2-CDR2-FR3-CDR3FR 4-constant region-stop codon heavy chain variable region (VH):

EVQLVESGGDLVKPGGSLKLSCAASGFTFSSGGMSWVRQTPDKRLEWVATISNGGGETYYEDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCAREEYEGTWYEDVWGAGTTVTVSS (SEQ ID NO:7) constant region

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT

ITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEF

KCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQ

PAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK-

Light chain:

FR1-CDR1-FR2-CDR2-FR3-CDR3FR 4-constant region-variable light chain region (VL) with stop codon

DVLMTQTPLSLPVSLGDQASISCTSSQSGVH

SNGNTELEWYLQKPGQSPKLLIYKVSNRTSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCGQGSHVG RTFGGGTKL

KEI(SEQ ID NO:8)

Constant region

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK

DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-

The RNA sequence encoding VH is presumed to be shown in SEQ ID NO. 12, and the RNA sequence encoding VL is presumed to be shown in SEQ ID NO. 13.

Example 55 affinity determination of binding of C12D2C8 antibody to antigen

The dissociation rate equilibrium constant (KD) of an antigen antibody may reflect the high or low affinity between the antibody and the antigen, with lower KD values leading to higher affinity. The affinity of a monoclonal antibody to an antigen was evaluated by measuring the antigen-antibody dissociation rate equilibrium constant of the monoclonal antibody using a Surface Plasmon Resonance (SPR) technique.

The binding curve of the SPR assay to antigen was performed on the monoclonal antibody 5C12D2C8 at various concentrations of 12.5, 25, 50, 100, 200, 400 nM. The results are shown in FIG. 5: the dissociation constant Kd (1/s) of monoclonal antibody 5C12D2C8 for antigen was 2.47x10^-4An equilibrium dissociation constant KD (M) of 8.78x10^-9(FIG. 4).

Example 65 specificity of the C12D2C8 antibody in human peripheral nerve cells

Since the nerve cells were highly differentiated, to examine the specificity of 5C12D2C8 in human peripheral nerve cells, verification was performed using iPS-induced human peripheral nerve cells. Before the test, the types of iPS-induced nerve cells are confirmed, peripheral nerve tissue specific molecular markers are adopted to identify whether the induced nerve cells belong to the neuron cells, and three molecules MarkerPSD95 specifically expressed in the neuron cells are used for positioning human nerve tissues. PSD95 is encoded by DLG4 gene, is one member of the membrane-associated guanylate kinase (MAGUK) family, and can interact with PSD93 at postsynaptic site and be specifically expressed in nervous tissue. Therefore, the PSD95 antibody can be used as a molecular Marker (control) to research the expression specificity and distribution of the antibody in human peripheral nerve cells.

6.1iPS Induction of human peripheral neurons

6.1.1: using 2. mu.g/cm²The lamin (SIGMA) -coated T25 flasks according to 2.5X10⁶Cell number per flask human neural stem cells (iRegene Therapeutics) were seeded in 2 coated T25 flasks in serum-free animal-derived component-free FP neural stem cell medium (iRegene Therapeutics) supplemented with 10. mu.M inhibitor Y-27632. The inoculated T25 culture flask was placed at 37 ℃ in 5% CO₂Cultured overnight in the medium.

6.1.2: the following day, the FP neural stem cell medium containing Y-27632 was removed and replaced with serum-free animal-derived component-free peripheral neuron directed differentiation medium (iRegene Therapeutics), and this medium was used until the end of the experiment. Then replacing the culture medium every other day until day 14; the morphological changes of the cells were recorded in the middle.

6.1.3: the bilayer was coated with 15mm optical slides (deckgalaser) to prepare cell crawlers. 15mm optical slides were placed in 24-well plates (1 plate/well). The slide was immersed in 50. mu.g/mL poly-L-lysine solution and placed at 37 ℃ in 5% CO₂The incubation was continued for not less than 2 hours. After removal of poly-L-lysine, it was washed 3 times with sterile water. The slides were immersed in 5. mu.g/mL Lamin solution and placed at 37 ℃ in 5% CO₂Incubate for not less than 3 hours, then use PBS to fully wash.

6.1.4: cells in T25 flasks were digested with Accutase cell digest (Invitrogen) at 1x10⁵Cell number per well cells were seeded on coated 15mm optical slides (deckgraser). The culture medium is serum-free and animal-derived component-free neuron directed differentiation medium (iRegene Therapeutics), and then the culture medium is replaced every other day until day 21.

6.2 fluorescent immunoassay

6.2.1: the medium for cell differentiation in the 24-well plates was removed and the culture wells were washed with DPBS.

6.2.2: fixing the cells with 4% paraformaldehyde at room temperature for 40 minutes, and washing twice with DPBS buffer solution; then treating the mixture with 0.1% Triton X-100 for 5 minutes, and washing the mixture twice with a DPBS buffer solution; cells were then incubated overnight at 4 ℃ with DPBS buffer containing 10% horse serum and 0.1% Triton X-100; finally adding the antibody diluted by the DPBS buffer solution, incubating for 2 hours at 37 ℃, and washing for three times by the DPBS buffer solution; then, a fluorescent secondary antibody (1:1000) corresponding to the primary antibody is used for incubation, and after 45 minutes, the secondary antibody is removed and washed for three times by using a DPBS buffer solution; nuclear staining was performed with 300nM DAPI for 2min at room temperature; cleaning the sealing sheet for three times by using sterilized water to prepare an optical cell climbing sheet; the slides were read using OLYMPUS FV3000 laser confocal microscope or NIKON-SIM high resolution microscope.

6.3 results of localization of human peripheral nerve cells

Cell type analysis is carried out on peripheral nerve cells induced by iPS by adopting a peripheral nerve cell specific molecular marker PSD95, and the result shows that: the peripheral nerve cells induced by iPS can be combined with specific molecular markers, so that the iPS-induced nerve cells belong to the peripheral nerve cells and can be used for cell specificity analysis of antibodies. The distribution of monoclonal antibody 5C12D2C8 on iPS-induced human peripheral nerve cells is shown in fig. 5: the monoclonal antibody 5C12D2C8 has stronger fluorescence signals on axons of peripheral nerve cells, and compared with a control group PSD95, the fluorescence intensity on the axons is stronger, and the result shows that the Nav1.7 channel monoclonal antibody 5C12D2C8 is co-located with the specific molecular markers of axon molecules, which indicates that the 5C12D2C8 has good peripheral nerve cell specificity and is in punctate arrangement on the axons of the peripheral nerve cells.

Example 75 analgesic Effect of the C12D2C8 antibody in wild-type mice

After nociceptors are stimulated, action potentials are generated and are transmitted to spinal cords through peripheral nerves and then transmitted to the brain to feel pain, Nav1.7 voltage-gated sodium ion channels play a crucial role in generation and transmission of the action potentials, and the monoclonal antibody drug specificity is supposed to be combined with ion transmission holes of the voltage-gated sodium ion channels Nav1.7 to possibly prevent electric signal transmission, so that the analgesic effect is achieved.

In order to verify the analgesic effect of monoclonal antibody 5C12D2C8, the formalin model of inflammatory pain was used in this study, and the pain caused by the formalin test was more biased towards the stressful pain and caused a state very similar to clinical pain, and this method has been widely used to determine the pain perception of animals. The behavior of the animal's response to noxious stimuli after formalin is presented as 2 phases, the first phase occurring immediately after injection for about 10min, the second phase occurring about 15min after injection for about 45min, and thus these 2 phases are designated as phase I and phase II, respectively. Phase I pain is believed to be caused by formalin directly stimulating nociceptors, causing C-fiber excitation to produce pain, while phase II pain is caused by inflammation causing the release of neurotransmitters such as prostaglandin, histamine, 5-hydroxytryptamine, etc., causing pain from pain nerve excitation, and phase II is believed to be more responsive to the action of drugs.

7.1 Experimental methods

7.1.1: an experimental instrument:

mouse tail injection fixator (YLS-Q9G) was purchased from Shanghai Soft-Longton scientific development Co., Ltd, and 50. mu.l microsyringe and 1ml human insulin syringe were purchased from Wuhan Shijie Jie.

7.1.2: experimental animals:

25-35g SPF-grade KM mice were purchased from the animal testing research center in Hubei province; KM mice were purchased at least in the laboratory to acclimate for about 2 days at ambient temperature of 23 + -1 deg.C, and given sufficient moisture and food, 5-8 mice per cage. Mice were acclimated in transparent cages for 30min prior to the experiment. Food and water are isolated in the experimental process, a timer is used for recording results, and the animals are placed into an ether poison bottle for anesthesia and euthanasia after the experiment is finished.

7.1.3: method for treating drugs

The mouse was fished out of the transparent cage, placed on a table top and pulled at its tail into the holder, and then the head was secured with a plug so that its tail extended out of the bottom of the holder. The fixator is placed on a tail injection auxiliary table, the tail of a mouse is placed under an auxiliary lamp for observation, 200 mu l of monoclonal antibody medicine is injected into the side blood vessel by a tail vein injection method after the side blood vessel is observed, the needle opening is pressed for 20s after the needle head is pulled out, and the mouse is placed into a transparent cage after hemostasis. 30min later, a micro-syringe is used to inject 20 mu l of 5% formalin solution which is freshly prepared into the soles of the mice, then the mice are immediately placed into a transparent cage for observation, the time(s) for the mice to lick the injection paw within 45min is recorded by a timer, data are recorded at intervals of 5min, the time for the mice to lick the paw and the paw is recorded within every 5min, the total time is 45min, the I-phase acute pain (0-10 min) and the II-phase persistent pain (10-45 min) are respectively analyzed in a statistical manner, and a control group is a mouse IgG control antibody with equal volume and equal concentration.

7.1.4: data processing

Carrying out double-tail T-test on the data to test whether the data have significance level, and carrying out double-tail T-test on experimental group data, a PBS control group and an IgG control group in the same dose experiment; in different dose experiments, the experimental group data of the drug was compared with the PBS control group and the IgG control group at equal doses, and different doses of the same drug were also compared. Significant differences are indicated by x, where x indicates p <0.05 and x indicates p < 0.01.

7.2 results of the experiment

The analgesic effect of monoclonal antibody 5C12D2C8 at a dose of 25mg/kg is shown in figure 6: the IgG negative antibody control (IgG) showed a significant increase in paw licking time in phase II compared to phase I at the same dose, whereas the injected monoclonal antibody 5C12D2C8 showed a very significant difference in paw licking time in phase II compared to phase I and control after administration (P <0.01), and the analgesic effect of monoclonal antibody 5C12D2C8 in phase II was reduced by 32% compared to the negative antibody control (IgG) (fig. 6).

Sequence listing:

HCDR 1:

SGGMS(SEQ ID NO:1)

HCDR2:

TISNGGGETYYEDSVKG(SEQ ID NO:2)

HCDR 3:

EEYEGTWYEDV(SEQ ID NO:3)

LCDR 1:

TSSQSGVHSNGNTELE(SEQ ID NO:4)

LCDR2:

KVSNRTS(SEQ ID NO:5)

LCDR 3:

GQGSHVGRT(SEQ ID NO:6)

VH:

MNFGLSLIFLALILKGVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFSSGGMSWVRQTPDKRLEWATISNGGGETYYEDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCAREEYEGTWYEDVWGAGTVTVSS(SEQ ID NO:7)

heavy chain:

MNFGLSLIFLALILKGVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFSSGGMSWVRQTPDKRLEWATISNGGGETYYEDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCAREEYEGTWYEDVWGAGT

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY

TLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT

ITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEF

KCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQ

PAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK-(SEQ ID NO：14)

VL：

MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCTSSQSGVHSNGNTELEWYLQKPGQPKLLIYKVSNRTSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCGQGSHVGRTFGGGTKLEI(SEQ ID NO:8)

light chain:

MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCTSSQSGVHSNGNTELEWYLQKPGQPKLLIYKVSNRTSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCGQGSHVGRTFGGGTKLEI

KRAAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS

TLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC- (SEQ ID NO: 15) antigenic peptide:

DSVNVDKQPKYEYS(SEQ ID NO:9)

VH nucleotide sequence:

atgaacttcgggctcagcttgattttccttgccctcattttaaaaggtgtccagtgtgaggtgcagc

tggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctgg

attcactttcagtagcggcggcatgtcttgggttcgccagactccagacaagaggctggagtgggtc

gcaaccattagtaatggtggtggtgagacctactatgaggacagtgtgaaggggcgattcaccatct

ccagagacaatgccaagaacaccctgtacctacaaatgaacagtctgaagtctgaggacacagccat

gtattactgtgctagggaggagtacgagggtacgtggtactgagatgtctggggcgcagggaccacg

gtcaccgtctcctca(SEQ ID NO:10)

heavy chain nucleotide sequence:

atgaacttcgggctcagcttgattttccttgccctcattttaaaaggtgtccagtgtgaggtgcagctggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtagcggcggcatgtcttgggttcgccagactccagacaagaggctggagtgggtcgcaaccattagtaatggtggtggtgagacctactatgaggacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctgtacctacaaatgaacagtctgaagtctgaggacacagccatgtattactgtgctagggaggagtacgagggtacgtggtactgagatgtctggggcgcagggaccacggtcaccgtctcctcagccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaatga(SEQ ID NO：16)

a VL nucleotide sequence:

atgaagttgcctgttaggctgttggtgctgatgttctggattcctgcttccagcagtgatgttttga

tgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcacatctag

tcagagcggagtacatagtaatggaaacaccgagttagaatggtacctgcagaaaccaggccagtct

ccaaagctcctgatctacaaagtttccaaccgaacatctggggtcccagacaggttcagtggcagtg

gatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactg

cggccaaggttcacatgttggccggacgttcggtggaggcaccaagctggaaatcaaa(SEQ ID NO:11)

the light chain nucleotide sequence:

atgaagttgcctgttaggctgttggtgctgatgttctggattcctgcttccagcagtgatgttttga

tgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcacatctag

tcagagcggagtacatagtaatggaaacaccgagttagaatggtacctgcagaaaccaggccagtct

ccaaagctcctgatctacaaagtttccaaccgaacatctggggtcccagacaggttcagtggcagtg

gatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactg

cggccaaggttcacatgttggccggacgttcggtggaggcaccaagctggaaatcaaacgggctgat

gctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcg

tgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacg

acaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagc

accctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaaga

catcaacttcacccattgtcaagagcttcaacaggaatgagtgttag(SEQ ID NO：17)

RNA sequence:

AUGAACUUCGGGCUCAGCUUGAUUUUCCUUGCCCUCAUUUUAAAAGGUGUCCAGUGUGAGGUGCAGCUGGUGGAGUCUGGGGGAGACUUAGUGAAGCCUGGAGGGUCCCUGAAACUCUCCUGUGCAGCCUCUGGAUUCACUUUCAGUAGCGGCGGCAUGUCUUGGGUUCGCCAGACUCCAGACAAGAGGCUGGAGUGGGUCGCAACCAUUAGUAAUGGUGGUGGUGAGACCUACUAUGAGGACAGUGUGAAGGGGCGAUUCACCAUCUCCAGAGACAAUGCCAAGAACACCCUGUACCUACAAAUGAACAGUCUGAAGUCUGAGGACACAGCCAUGUAUUACUGUGCUAGGGAGGAGUACGAGGGUACGUGGUACUGAGAUGUCUGGGGCGCAGGGACCACGGUCACCGUCUCCUCA(SEQ ID NO：12)

AUGAAGUUGCCUGUUAGGCUGUUGGUGCUGAUGUUCUGGAUUCCUGCUUCCAGCAGUGAUGUUUUGAUGACCCAAACUCCACUCUCCCUGCCUGUCAGUCUUGGAGAUCAAGCCUCCAUCUCUUGCACAUCUAGUCAGAGCGGAGUACAUAGUAAUGGAAACACCGAGUUAGAAUGGUACCUGCAGAAACCAGGCCAGUCUCCAAAGCUCCUGAUCUACAAAGUUUCCAACCGAACAUCUGGGGUCCCAGACAGGUUCAGUGGCAGUGGAUCAGGGACAGAUUUCACACUCAAGAUCAGCAGAGUGGAGGCUGAGGAUCUGGGAGUUUAUUACUGCGGCCAAGGUUCACAUGUUGGCCGGACGUUCGGUGGAGGCACCAAGCUGGAAAUCAAA(SEQ ID NO：13)

SEQUENCE LISTING

<110> Yisenhui (Wuhan) biopharmaceutical Co., Ltd

<120> antibody or antibody fragment specifically binding to voltage-gated sodium ion channel alpha subunit Nav1.7

<130> WH1917-20P150058

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Mus musculus

<400> 1

Ser Gly Gly Met Ser

1 5

<210> 2

<211> 17

<212> PRT

<213> Mus musculus

<400> 2

Thr Ile Ser Asn Gly Gly Gly Glu Thr Tyr Tyr Glu Asp Ser Val Lys

1 5 10 15

Gly

<210> 3

<211> 11

<212> PRT

<213> Mus musculus

<400> 3

Glu Glu Tyr Glu Gly Thr Trp Tyr Glu Asp Val

1 5 10

<210> 4

<211> 16

<212> PRT

<213> Mus musculus

<400> 4

Thr Ser Ser Gln Ser Gly Val His Ser Asn Gly Asn Thr Glu Leu Glu

1 5 10 15

<210> 5

<211> 7

<212> PRT

<213> Mus musculus

<400> 5

Lys Val Ser Asn Arg Thr Ser

1 5

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<212> PRT

<213> Mus musculus

<400> 6

Gly Gln Gly Ser His Val Gly Arg Thr

1 5

<210> 7

<211> 137

<212> PRT

<213> Synthetic Protein

<400> 7

Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Ala Leu Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Gly Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu

50 55 60

Glu Trp Ala Thr Ile Ser Asn Gly Gly Gly Glu Thr Tyr Tyr Glu Asp

65 70 75 80

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

85 90 95

Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr

100 105 110

Tyr Cys Ala Arg Glu Glu Tyr Glu Gly Thr Trp Tyr Glu Asp Val Trp

115 120 125

Gly Ala Gly Thr Val Thr Val Ser Ser

130 135

<210> 8

<211> 129

<212> PRT

<213> Synthetic Protein

<400> 8

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Thr Ser Ser Gln Ser Gly

35 40 45

Val His Ser Asn Gly Asn Thr Glu Leu Glu Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Thr Ser Gly

65 70 75 80

Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Gly

100 105 110

Gln Gly Ser His Val Gly Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile

<210> 9

<211> 14

<212> PRT

<213> Synthetic Protein

<400> 9

Asp Ser Val Asn Val Asp Lys Gln Pro Lys Tyr Glu Tyr Ser

1 5 10

<210> 10

<211> 417

<212> DNA

<213> Synthetic

<400> 10

atgaacttcg ggctcagctt gattttcctt gccctcattt taaaaggtgt ccagtgtgag 60

gtgcagctgg tggagtctgg gggagactta gtgaagcctg gagggtccct gaaactctcc 120

tgtgcagcct ctggattcac tttcagtagc ggcggcatgt cttgggttcg ccagactcca 180

gacaagaggc tggagtgggt cgcaaccatt agtaatggtg gtggtgagac ctactatgag 240

gacagtgtga aggggcgatt caccatctcc agagacaatg ccaagaacac cctgtaccta 300

caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgctag ggaggagtac 360

gagggtacgt ggtactgaga tgtctggggc gcagggacca cggtcaccgt ctcctca 417

<210> 11

<211> 393

<212> DNA

<213> Synthetic

<400> 11

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60

gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcacat ctagtcagag cggagtacat agtaatggaa acaccgagtt agaatggtac 180

ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgaacatct 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat tactgcggcc aaggttcaca tgttggccgg 360

acgttcggtg gaggcaccaa gctggaaatc aaa 393

<210> 12

<211> 417

<212> RNA

<213> Synthetic

<400> 12

augaacuucg ggcucagcuu gauuuuccuu gcccucauuu uaaaaggugu ccagugugag 60

gugcagcugg uggagucugg gggagacuua gugaagccug gagggucccu gaaacucucc 120

ugugcagccu cuggauucac uuucaguagc ggcggcaugu cuuggguucg ccagacucca 180

gacaagaggc uggagugggu cgcaaccauu aguaauggug guggugagac cuacuaugag 240

gacaguguga aggggcgauu caccaucucc agagacaaug ccaagaacac ccuguaccua 300

caaaugaaca gucugaaguc ugaggacaca gccauguauu acugugcuag ggaggaguac 360

gaggguacgu gguacugaga ugucuggggc gcagggacca cggucaccgu cuccuca 417

<210> 13

<211> 393

<212> RNA

<213> Synthetic

<400> 13

augaaguugc cuguuaggcu guuggugcug auguucugga uuccugcuuc cagcagugau 60

guuuugauga cccaaacucc acucucccug ccugucaguc uuggagauca agccuccauc 120

ucuugcacau cuagucagag cggaguacau aguaauggaa acaccgaguu agaaugguac 180

cugcagaaac caggccaguc uccaaagcuc cugaucuaca aaguuuccaa ccgaacaucu 240

ggggucccag acagguucag uggcagugga ucagggacag auuucacacu caagaucagc 300

agaguggagg cugaggaucu gggaguuuau uacugcggcc aagguucaca uguuggccgg 360

acguucggug gaggcaccaa gcuggaaauc aaa 393

<210> 14

<211> 456

<212> PRT

<213> Synthetic

<400> 14

Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Ala Leu Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Gly Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu

50 55 60

Glu Trp Ala Thr Ile Ser Asn Gly Gly Gly Glu Thr Tyr Tyr Glu Asp

65 70 75 80

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

85 90 95

Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr

100 105 110

Tyr Cys Ala Arg Glu Glu Tyr Glu Gly Thr Trp Tyr Glu Asp Val Trp

115 120 125

Gly Ala Gly Thr Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala

130 135 140

Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu

145 150 155 160

Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly

165 170 175

Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp

180 185 190

Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro

195 200 205

Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys

210 215 220

Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile

225 230 235 240

Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro

245 250 255

Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val

260 265 270

Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val

275 280 285

Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln

290 295 300

Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln

305 310 315 320

Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala

325 330 335

Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro

340 345 350

Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala

355 360 365

Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu

370 375 380

Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr

385 390 395 400

Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr

405 410 415

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

420 425 430

Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys

435 440 445

Ser Leu Ser His Ser Pro Gly Lys

450 455

<210> 15

<211> 236

<212> PRT

<213> Synthetic

<400> 15

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Thr Ser Ser Gln Ser Gly

35 40 45

Val His Ser Asn Gly Asn Thr Glu Leu Glu Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Thr Ser Gly

65 70 75 80

Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Gly

100 105 110

Gln Gly Ser His Val Gly Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys Arg Ala Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser

130 135 140

Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn

145 150 155 160

Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu

165 170 175

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

180 185 190

Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr

195 200 205

Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr

210 215 220

Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 16

<211> 1392

<212> DNA

<213> Synthetic

<400> 16

atgaacttcg ggctcagctt gattttcctt gccctcattt taaaaggtgt ccagtgtgag 60

gtgcagctgg tggagtctgg gggagactta gtgaagcctg gagggtccct gaaactctcc 120

tgtgcagcct ctggattcac tttcagtagc ggcggcatgt cttgggttcg ccagactcca 180

gacaagaggc tggagtgggt cgcaaccatt agtaatggtg gtggtgagac ctactatgag 240

gacagtgtga aggggcgatt caccatctcc agagacaatg ccaagaacac cctgtaccta 300

caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgctag ggaggagtac 360

gagggtacgt ggtactgaga tgtctggggc gcagggacca cggtcaccgt ctcctcagcc 420

aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc 480

atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg 540

aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc 600

tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc 660

tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat 720

tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc 780

ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta 840

gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg 900

cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt 960

gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac 1020

agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag 1080

gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt 1140

ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat 1200

gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac 1260

ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc 1320

tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct 1380

cctggtaaat ga 1392

<210> 17

<211> 717

<212> DNA

<213> Synthetic

<400> 17

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60

gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcacat ctagtcagag cggagtacat agtaatggaa acaccgagtt agaatggtac 180

ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgaacatct 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat tactgcggcc aaggttcaca tgttggccgg 360

acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc 420

atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480

aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540

aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600

agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660

actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag 717

Claims

1. An antibody or antibody fragment that specifically binds to voltage-gated sodium ion channel alpha subunit Nav1.7, wherein the target to which the antibody or antibody fragment specifically binds is an ion-conducting pore module of the DIVS3 domain of the voltage-gated sodium ion channel alpha subunit.

2. The antibody or antibody fragment of claim 1, characterized in that: the antibody or antibody fragment comprises:

3. The antibody or antibody fragment thereof according to any one of claims 1-2, characterized in that said antibody or antibody fragment comprises the heavy chain variable region as set forth in SEQ ID No.7 and/or the light chain variable region as set forth in SEQ ID No. 8.

4. The antibody or antibody fragment thereof according to any one of claims 1 to 3, characterized in that the antibody further comprises an antibody constant region.

5. The antibody or antibody fragment according to any one of claims 1 to 4, which antibody or antibody fragment comprises a heavy chain as shown in SEQ ID No.14 and/or a light chain as shown in SEQ ID No. 15.

6. The antibody or antibody fragment thereof according to claim 1, characterized in that said antibody or antibody fragment is selected from the following structural forms: whole antibody, Fab, F (ab')₂dsFv, scFv, diabodies, minibodies, diabodies, multispecific antibodies, chimeric antibodies and CDR-grafted antibodies.

7. The antibody or antibody fragment thereof according to claim 1, characterized in that the antibody is a monoclonal antibody.

8. The antibody or antibody fragment thereof according to claim 7, characterized in that the antibody is a humanized antibody.

9. A polypeptide which specifically binds to the antibody or antibody fragment of claim 1, characterized in that said polypeptide has the amino acid sequence shown in SEQ ID No. 9.

10. An isolated nucleotide encoding an antibody or antibody fragment of any one of claims 1-8 that specifically binds to the α subunit Nav1.7 of a voltage-gated sodium ion channel.

11. An expression vector comprising the nucleotide of claim 10.

12. A host cell containing the nucleotide of claim 10 or the expression vector of claim 11.

13. A method of producing an antibody or antibody fragment according to claim 1 that specifically binds to the α subunit nav1.7 of a voltage-gated sodium ion channel, said method comprising the steps of:

(a) culturing the host cell of claim 12 under expression conditions such that the antibody or antibody fragment that specifically binds to voltage-gated sodium ion channel α subunit nav1.7 is expressed;

14. A pharmaceutical composition comprising the antibody or antibody fragment of any one of claims 1 to 8.

15. Use of an antibody or antibody fragment according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 14 for the manufacture of a medicament for the treatment of a pain-related disorder.