CN112724253B

CN112724253B - Antibody of anti-human vault protein and application thereof

Info

Publication number: CN112724253B
Application number: CN202110257408.7A
Authority: CN
Inventors: 付康; 赵逸堃; 李胡丹
Original assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Current assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Priority date: 2021-03-09
Filing date: 2021-03-09
Publication date: 2022-04-05
Anticipated expiration: 2041-03-09
Also published as: CN112724253A

Abstract

The invention discloses an antibody of an anti-human vault protein and application thereof, and relates to the technical field of biological medicines. The antibody disclosed by the invention comprises a heavy chain variable region shown in SEQ ID NO.1 and a light chain variable region shown in SEQ ID NO. 2. The antibody can be specifically combined with human fornix protein, has good specificity and higher affinity, and can be used for diagnosing related diseases taking the fornix protein as a marker and detecting the human fornix protein.

Description

Antibody of anti-human vault protein and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to an antibody for resisting human vault protein and application thereof.

Background

Tumor multidrug resistance (MDR) is one of the troubles in the research of tumor therapy in recent years, and the generation mechanism is very complicated. With the continuous and intensive research on the mechanism of non-P-glycoprotein mediated MDR, lung resistance-related protein (LRP) is proved to play an important role in DMR and is expected to become a new therapeutic target. The LRP gene has been cloned successfully by Scheper et al, and the gene is identical to the sequence of 87% of the amino acid of the MVP of the limousine, so that the LRP is deduced to be human fornix protein. The vault protein, also known as MVP. Through research, a drug-resistant cell strain is screened from a cell strain of human non-small cell lung cancer, and the amino acid series of the drug-resistant cell strain has 87.7 percent of homology with vault protein (MVP), so the MVP is also called lung drug-resistant related protein.

Fornices are important organelles, widely distributed in many species, highly conserved in eukaryotes, mostly cytoplasmic, 5% linked to the nuclear membrane near the nuclear pore. LRP in different cell types was uniquely localized to the cytosol, in rough granules or vesicles, observed by focusing microscopy, and not seen in nuclear pore complexes.

The phenomenon of multidrug resistance (MDR) in tumors is a major problem that plagues clinical treatment, and the relationship between the fornix and MDR has been extensively studied after the lung drug resistance protein has been identified as MVP. Many studies have demonstrated that the expression intensity of MVP in MDR cells is positively correlated with the drug resistance, and the protein level of MVP is up-regulated to different degrees. Meanwhile, by counting 86 cases of acute lymphoblastic leukemia, MVP is highly expressed in relapsers, and the positive rate is 85.7%.

Because of the relevance of LRP and the multidrug resistance of tumor cells, both domestic and foreign studies report that MVP is used as a treatment and prognosis evaluation index by analyzing the relation between the expression of MVP and the biological behavior and prognosis index of tumor patients. Pulmonary drug resistance-related proteins produce drug resistance primarily by preventing drugs from entering the nucleus or drugs that enter the cytoplasm enter the transport vesicle, are excreted exocellularly to reduce intracellular drug concentrations. If the MVP is highly expressed, poor prognosis is suggested. The anti-MVP monoclonal antibody will facilitate the diagnosis of cancer therapy and prognosis and make the diagnosis of cancer independent of etiology, and therefore more convenient. However, monoclonal antibodies against MVP are currently lacking.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The antibody of the anti-human vault protein provided by the invention can be specifically combined with the human vault protein, has good specificity and higher affinity, and can be used for diagnosing related diseases taking the vault protein as a marker and detecting the human vault protein.

The invention is realized by the following steps:

in one aspect, the invention provides an antibody against human vault protein or an antigen binding fragment thereof, wherein the amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID No.1, and the amino acid sequence of a light chain variable region of the antibody is shown as SEQ ID No. 2.

The antibody of the anti-human vault protein provided by the invention has the heavy chain variable region and the light chain variable region of the amino acid sequence, can specifically recognize the human vault protein, can be used for detecting the human vault protein, can also be used as a diagnostic reagent for diagnosing related diseases such as tumors and the like which take the vault protein as a marker, and has higher specificity and affinity for the human vault protein.

The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, "antibody" includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab ', F (ab')2, Fv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified arrangement of immunoglobulin molecules comprising an antigen recognition site of a desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies, among others.

Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (VH) and first, second and third constant regions (CH1, CH2 and CH 3). Each light chain contains a light chain variable region (VL) and a constant region (CL). Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but the antibodies need not belong to any particular class. Antibodies can be classified into different classes according to the antibody amino acid sequence of the constant region of the heavy chain. Generally, there are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant regions corresponding to different antibody classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of antibodies are well known.

Alternatively, in some embodiments of the invention, the antigen binding fragment is selected from any one of Fab ', Fab, F (ab')2, scFv and Fv of the antibody.

The term "antigen-binding fragment," as used herein, refers to a portion or region of an intact antibody molecule that is responsible for binding to an antigen (e.g., a human vault protein or a complex containing a human vault protein). It may comprise the heavy chain variable region (VH), the light chain variable region (VL), or both, typically in the form of Fab ', Fab, F (ab')2, scFv, and Fv structures, which are well known to those skilled in the art.

Alternatively, in some embodiments of the invention, the constant region of the antibody is selected from the constant regions of any one of IgG, IgA, IgM, IgE and IgD.

Alternatively, in some embodiments of the invention, the species source of the constant region of the antibody is mouse, rat, bovine, horse, sheep, rabbit or dog.

Alternatively, in some embodiments of the invention, the species source of the constant region of the antibody is a mouse.

It should be noted that, those skilled in the art can make various modifications such as humanization modification, affinity modification, etc. to the antibody provided by the present invention as needed, and any modifications may be made, so long as the antibody obtained by modifying the antibody provided by the present invention on the basis of the variable region sequence described above or the complementarity determining region thereof is within the scope of the present invention.

On the other hand, the invention also provides the application of the antibody or the antigen binding fragment thereof in preparing a tumor diagnostic reagent or a kit, wherein the diagnostic marker of the tumor is human fornix protein.

Tumors with human vault proteins as markers include: lung adenocarcinoma, breast cancer, and the like.

In another aspect, the invention also provides an immunoconjugate, bispecific molecule, chimeric antigen receptor or pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above.

In another aspect, the present invention also provides a detection kit comprising the antibody or antigen-binding fragment thereof as described above.

Alternatively, in some embodiments of the invention, when the antibody or antigen-binding fragment thereof provided herein is used as a detection reagent to detect human vault protein, the antibody or antigen-binding fragment thereof has a detectable label.

The detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes, but is not limited thereto. When an enzyme is used as the detection label, the enzyme includes, but is not limited to, β -glucuronidase, β -D-glucosidase, β -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase, guanosine diphosphate (GDPase), ribonuclease, glucose oxidase, luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, β -lactamase, and the like. When a fluorescent material is used as the detection label, the fluorescent material includes, but is not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, and the like. When a ligand is used as the detection label, the ligand includes, but is not limited to, biotin derivatives and the like. When a light-emitting material is used as the detection label, the light-emitting material includes, but is not limited to, acridinium ester, luciferin, luciferase, and the like. When microparticles are used as the detection label, the microparticles include, but are not limited to, colloidal gold, colored latex, and the like. When a redox molecule is used as the detection label, the redox molecule includes, but is not limited to, ferrocene, ruthenium complexes, viologen, quinone, Ti ion, Cs ion, diimide, 1, 4-benzoquinone, hydroquinone, K₄W(CN)₈、[Os(bpy)₃]²⁺、[RU(bpy)₃]²⁺、[MO(CN)₈]^4-And the like. When a radioisotope is used as the detection marker, the radioisotope includes, but is not limited to³H、¹⁴C、³²P、³⁵S、³⁶Cl、⁵¹Cr、⁵⁷Co、⁵⁸Co、⁵⁹Fe、⁹⁰Y、¹²⁵I、¹³¹I and¹⁸⁶re, and the like.

In another aspect, the invention also provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described above.

Alternatively, in some embodiments of the invention, the nucleic acid sequence of the nucleic acid molecule encoding the heavy chain variable region described above is as set forth in SEQ ID NO. 3.

Alternatively, in some embodiments of the invention, the nucleic acid sequence of the nucleic acid molecule encoding the light chain variable region is as set forth in SEQ ID NO. 4.

Based on the common general knowledge in the art, for example, according to the codon relationship corresponding to the amino acids shown in the following table, the skilled person can easily obtain the nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof according to the amino acid sequence provided by the present invention.

In another aspect, the present invention also provides a recombinant vector comprising a nucleic acid molecule as described above.

In another aspect, the present invention also provides a recombinant cell comprising the recombinant vector as described above.

In another aspect, the present invention also provides a method for preparing the antibody or the antigen-binding fragment thereof, comprising culturing the recombinant cell, and separating and purifying the cultured product to obtain the antibody or the antigen-binding fragment thereof.

Based on the above-mentioned antibody sequences provided by the present invention, those skilled in the art can obtain the above-mentioned antibody or antigen-binding fragment of the present invention by a conventional technique in the art, such as genetic engineering techniques, and the like, and the above-mentioned antibody of the present invention can be produced by any method, and thus, it falls within the scope of the present invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a picture of positive cells in the Immunocytochemistry (ICC) screening of example 1.

FIG. 2 shows bands of interest of the heavy and light chains of the monoclonal cell line 2D6-1 in example 2 after pcr amplification on agarose gel electrophoresis.

FIG. 3 shows the results of staining in immunohistochemistry after using cell supernatant of MVP monoclonal cell line (2D6-1) as a primary antibody in example 3.

FIG. 4 shows the results of the staining of the cell supernatant obtained by neutralization reaction with antigen and immunohistochemistry as a primary antibody using the MVP monoclonal cell line (2D6-1) in example 3.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Obtaining of mouse monoclonal antibody with specificity to MVP

1 animal (mouse) immunization

Mice are immunized with the immunogen according to methods commonly used in the literature (E Harlow, D.Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998). The immunogen is recombinant human MVP protein (the amino acid sequence is shown as SEQ ID NO. 5) with a His label, and is used as a detection antigen to measure and screen serum titer and hybridoma, the high-purity antigen can increase the chance of obtaining the required monoclonal antibody, and the screening amount is reduced. 5 mice were immunized, each mouse being immunized with 50 μ g of MVP antigen. The antigen protein solution was prepared with PBS. Proper amount of antigen protein, PBS and Freund's adjuvant are put into an injector, the water outlet of the injector is plugged by a plug, and the injector is put on an emulsifying instrument to be fully stirred and emulsified, so that the antigen adjuvant forms stable water-in-oil solution. The first tail blood serum titer test is carried out 7-10 days after the primary and secondary immunization, and good titer is obtained after 2-4 times of boosting immunization. And finally, the immune mice with high serum titer are subjected to cell fusion after abdominal cavity immunization.

The amino acid sequence of the recombinant human MVP protein is as follows:

MATEEFIIRIPPYHYIHVLDQNSNVSRVEVGPKTYIRQDNERVLFAPMRMVTVPPRHYCTVANPVSRDAQGLVLFDVTGQVRLRHADLEIRLAQDPFPLYPGEVLEKDITPLQVVLPNTALHLKALLDFEDKDGDKVVAGDEWLFEGPGTYIPRKEVEVVEIIQATIIRQNQALRLRARKECWDRDGKERVTGEEWLVTTVGAYLPAVFEEVLDLVDAVILTEKTALHLRARRNFRDFRGVSRRTGEEWLVTVQDTEAHVPDVHEEVLGVVPITTLGPHNYCVILDPVGPDGKNQLGQKRVVKGEKSFFLQPGEQLEQGIQDVYVLSEQQGLLLRALQPLEEGEDEEKVSHQAGDHWLIRGPLEYVPSAKVEVVEERQAIPLDENEGIYVQDVKTGKVRA。

2 hybridoma cell fusion and screening

Preparation is required prior to cell fusion: (1) culturing mouse myeloma cells SP2/0 to logarithmic growth phase; (2) killing a negative mouse one day before fusion, injecting HAT culture medium into the abdominal cavity of the mouse in a sterile environment, taking the abdominal cavity trophoblast cells of the mouse, and paving the cells on a 96-well plate, wherein each well is 100 mu l, and the cells have a promoting effect on the growth of hybridoma cells; (3) the immunized mice were sacrificed, spleens were removed under sterile conditions, splenic B cells and SP2/0 myeloma cells were chemically fused using PEG according to the method described in the above document, and appropriate HAT medium was added according to the number of plates to be plated; (4) finally, the fused cells were plated on trophoblast cell culture plates at 100. mu.l per well.

After 7-10 days, the growth of viable hybridoma cells can be observed under a microscope, and after two weeks of plating, the supernatants of each well are collected and screened for hybridoma cells using the human MVP-his protein antigen by ELISA. The method comprises the following steps: the microplate was coated with 100. mu.l of 2. mu.g/ml PBS solution of human MVP-his protein antigen for two hours at 37 ℃. After PBST washing 3 times, add 150 u l/hole 3% skim milk powder PBS solution to 4 degrees C closed overnight. The plate was washed three more times, 80. mu.l/well of hybridoma supernatant was added, incubated at 37 ℃ for 1 hour, and then washed three times. Add 1: 7000 dilution of horseradish peroxidase labeled goat anti-mouse secondary antibody, 37 degrees C were incubated for 45 minutes, plate 3 times, dried. After 100. mu.l/well of TMB developing solution was added, development was carried out at room temperature for 5 to 10 minutes, and the reaction was stopped with 2M sulfuric acid solution, and the absorbance of each well at 450nm was measured. And (4) selecting positive hybridoma cells.

ELISA positive fusion wells were selected and then immunocytochemical staining (ICC) was performed, and positive wells were selected for subsequent experiments. The experimental procedure was as follows: (1) the adherent cell human non-small cell lung cancer cell (A549) is well digested by pancreatin digestive juice and then is paved in a 96-well plate in a few days before the experiment, and the plate is paved after the adherent growth; (2) taking out the 96-well plate, pouring out the culture medium, and washing with PBS for 3 times; (3) adding 4% paraformaldehyde, fixing at room temperature for 15-30 minutes, and washing the plate with PBS for three times; (4) 0.2% triton x-100 for 10-15min at room temperature, rinsing three times with PBS; (5) treating with 3% hydrogen peroxide for 15min, and rinsing with PBS for 3 times; 6.3% goat serum was blocked at 37 ℃ for 1 hour; (6) 80. mu.l of fusion well ELISA positive cell supernatant was added to the icc assay plate overnight at 4 ℃; (7) after PBST rinsing, adding a secondary antibody for reacting for 40min at room temperature; (8) DAB color development; (9) staining with hematoxylin; (10) PBST was observed in reverse blue, see FIG. 1.

The fusion cells which are combined to be positive are selected through ELISA and ICC experiments, are cloned through a limiting dilution method, and each positive strain is laid on an 48/96 pore plate and is continuously cultured. And performing a second round of re-screening by an ELISA method, screening out hybridomas which specifically recognize MVP and can block the combination of MVP, and subcloning by a limiting dilution method to obtain a monoclonal cell strain (2D 6-1). Expanding the monoclonal cell line, collecting about 1X 10⁶Injecting the selected mouse with one week of paraffin oil, allowing the mouse to generate ascites after 7-10 days, and collecting ascites for antibody purification. After purification, a mouse monoclonal antibody with specificity against human MVP is obtained.

Example 2

DNA cloning and sequencing, and variable region gene sequencing of the antihuman MVP monoclonal antibody.

Total RNA was extracted from mouse monoclonal cell lines using Trizol reagent. Cells cultured in 9cm dishes were collected into 1.5ml centrifuge tubes and the supernatant was blotted dry. Adding 1ml of Trizol reagent, blowing the cells evenly to crack the cells, and placing the cracked sample or homogenate at room temperature for 5-10min to completely separate the nucleoprotein and the nucleic acid. 0.2ml of chloroform was added thereto, followed by vigorous shaking for 15sec and standing at room temperature for 3 min. Centrifuge at 12000rpm4 ℃ for 10 min. Absorb the supernatantTransferring the phase to a clean centrifuge tube, adding isopropanol with the same volume, mixing uniformly, and standing at room temperature for 20 min. Centrifuge at 12000rpm for 10min at 4 deg.C, and discard the supernatant. The precipitate was washed by adding 1ml of 75% ethanol. Centrifuge at 12000rpm for 3min at 4 ℃ and discard the supernatant. Drying at room temperature for 5-10 min. Adding 30-50 μ l RNase-free ddH₂And O. The resulting RNA solution was stored at-70 ℃ or used for subsequent experiments.

The AMV first strand cDNA synthesis kit is selected to reverse transcribe total RNA into cDNA. The experimental system was configured as follows, 6. mu.l total RNA + 1. mu.l Oligo dT + 4. mu.l RNase-free water (total 11. mu.l). After gentle mixing, the mixture was centrifuged for 3-5s, and after the reaction mixture was pre-denatured by 5mi in a bath at 65 ℃, it was ice-cooled for 30s, then centrifuged for 3-5s, followed by ice-cooling for 2 min. Mu.l of 5 Xbuffer solution + 1. mu.l of dNTP mixture + 1. mu.l of RNase inhibitor + 1. mu.l of reverse transcriptase (20. mu.l of system in total) were added in an ice bath, gently mixed and centrifuged for 3 to 5 seconds at 42 ℃ for 50 minutes and 85 ℃ for 5 minutes in a PCR apparatus to complete the cDNA synthesis. The random primer is suitable for synthesizing short-chain cDNA with the length of less than 500bp, and the transcribed RNA template can be transcribed into a 5' end region without a poly (A) tail.

PCR amplification of light and heavy chains. For the sequence of the variable region of the amplified antibody light chain, a PCR reaction system is configured: 2 XTaq enzyme buffer 25. mu.l + FP-VL 1. mu.l + RP-VL 1. mu.l + cDNA 2. mu.l + ddH₂O21. mu.l. For the amplification of the heavy chain variable region sequence of the antibody, a PCR reaction system is configured: 2 XTaq enzyme buffer 25. mu.l + FP-VH 1. mu.l + RP-VH 1. mu.l + cDNA 2. mu.l + ddH₂O21. mu.l. The temperature cycles for PCR amplification of the heavy and light chain variable regions were as follows (with steps 2 to 4, repeated for 35 cycles):

step 1-pre-denaturation at 94 ℃ for 4 min;

step 2-denaturation at 94 ℃ for 30 sec;

step 3-annealing at 55 ℃ for 45 sec;

step 4-extension 72 ℃ for 60 sec;

step 5-72 ℃, 10 min;

step 6-store 4 ℃.

The PCR products were analyzed by 1% agarose gel electrophoresis (see FIG. 2), bands of DNA segments of corresponding sizes (about 375bp for VH and about 325bp for VL) were excised, and DNA extraction was performed using a DNA gel recovery kit of the SanPrep column type. Briefly described as follows: cutting off a gel block containing the target fragment from the agarose gel, and weighing; adding buffer B2 with the weight 3-6 times of the weight of the glue block, and carrying out water bath at 50 ℃ for 5-10 minutes to obtain sol; transferring the sol solution into an adsorption column, and centrifuging at 8000g for 30 s; pouring out the liquid in the collecting pipe; adding 500 μ l wash Solution into the column, centrifuging at 9000g for 30s, and pouring off the liquid in the collecting tube; adding wash solution repeatedly for one time, and pouring out the liquid; the pore adsorption column was centrifuged at 9000g for 1 min; the adsorption column was placed in a clean 1.5ml centrifuge tube, 15-40. mu.l of Elution Buffer was added to the center of the adsorption membrane, and after standing at room temperature for 1 minute, the column was centrifuged for 1 minute. Obtaining the prepared DNA solution, purifying PCR products and obtaining the variable region sequence of the antibody through sequencing. The sequencing results are shown in Table 1 below.

TABLE 1 amino acid sequences of VH and VL of murine anti-human MVP monoclonal antibody secreted by monoclonal cell line 2D6-1

Example 3

Immunohistochemistry (IHC) -based determination of the binding Capacity of an antibody to Lung drug resistance-related proteins

Baking the breast cancer tissue slices in a constant temperature box at 60 ℃ for 60 minutes, soaking the slices in xylene I for 15 minutes, replacing the xylene II, soaking for 15 minutes, soaking in absolute ethyl alcohol for 5 minutes, soaking in 95% ethanol for 5 minutes, soaking in 85% ethanol for 5 minutes, soaking in 75% ethanol for 5 minutes, and soaking in ddH₂Soaking for 5 minutes in O, and cleaning for 3 times; adding 10mmol/L citrate buffer solution (pH6.0) into a pressure cooker for antigen retrieval (boiling method), heating to boil, placing the slices on a heat-resistant material slicing frame, placing into the pressure cooker, covering the pressure cooker cover, covering the pressure valve, heating, maintaining the pressure for 4min, opening the vent valve to release gas after the time is up, opening the pressure cooker cover after the pressure is zero, taking out the inner cooker, and cooling at room temperature. Cooling the solution to room temperature, and collectingSlicing (about 40 min); ddH₂Soaking for 5 minutes in O, washing for 2 times, soaking for 5 minutes in PBST, and washing for 2 times; place the sections in 20ml 3% H₂O₂-in methanol solution, protected from light, at room temperature for 10 minutes; PBST is soaked for 5 minutes and washed for 3 times; adding one drop of goat serum blocking solution to each tissue group, wherein the drop is about 25 mu l, and incubating for 45 minutes at room temperature in a wet box; PBST was soaked for 5 minutes and washed 3 times. The cell supernatant of the MVP monoclonal cell line (2D6-1) of example 1 was added to each tissue and incubated overnight in a wet box at 4 ℃; taking out from a refrigerator at 4 ℃, and incubating for 60 minutes at room temperature; PBST is soaked for 5 minutes after being washed softly, and is washed for 3 times; 25. mu.l of HRP-labeled secondary antibody (CAT #: Inc.) was added to each tissue group, and the mixture was incubated at room temperature for 45 minutes; washing; preparing DAB color development liquid, reacting for 10-15 minutes in a dark place, and then dropwise adding the DAB color development liquid onto the slices for developing for 1-5 minutes; terminating the color reaction with distilled water; dripping 50 mul hematoxylin staining solution into each tissue group, staining for 5-10min, and washing with distilled water; placing the slices into 1% hydrochloric acid-ethanol for decoloring for 2-3 seconds, quickly taking out the slices, placing the slices into distilled water for stopping, and then placing the slices into PBST (pH8.0) for anti-blue for 5-10 minutes; soaking in 75% ethanol for 5 min: soaking in 85% ethanol for 5 min; soaking in 95% ethanol for 5 minutes: soaking in anhydrous ethanol for 5 min. Soaking in xylene for 10min, and soaking for 10min after xylene exchange; dripping neutral gum sealing sheet, and covering with glass slide; the microscope was photographed as shown in fig. 3.

Immunohistochemical neutralization experiments: a certain amount of antigen protein was mixed with cell supernatant of MVP monoclonal cell strain (2D6-1), and neutralized at 37 ℃ for 1 hour. Following the immunohistochemical protocol, as a single anti-drop, the breast cancer tissue sections were incubated overnight at 4 ℃ and then immunohistochemically stained and photographed following the same protocol, as shown in FIG. 4.

As can be seen from fig. 4, in the immunohistochemical neutralization reaction, the MVP antigen protein reaction binds to the antibody in the cell supernatant, so that the antibody in the cell supernatant cannot bind to the antigen in the cancer tissue. In fig. 3, it can be seen that the anti-human MVP monoclonal antibody 2D6-1 provided in the present invention can recognize and bind to MVP protein in human cancer cells, so that cancer cell cytoplasm in breast cancer tissue is brown. Illustrating that the anti-human MVP monoclonal antibody provided in example 1 can specifically bind to MVP.

Example 4

Affinity and sensitivity detection of antibodies

(1) The antigen was plated at 3mg/L, 1.5mg/L, 0.75mg/L, 0.375 mg/L.

(2) The antibody concentration of example 1 was adjusted to a level of 10-7mol/L (any of 1X 10-7 to 5X 10-7 mol/L).

(3) Then diluted 1:2-1:256 in multiple ratios and added to wells of different antigen coating amounts. Adding secondary antibody, developing TMB, and measuring the absorbance at 450 nm. The results are shown in Table 2.

TABLE 2

As can be seen from Table 2, the anti-human MVP monoclonal antibody of example 1 was able to detect as low as 0.375mg/L antigen, indicating its higher sensitivity.

(4) According to the antigen-antibody combination S curve, the antibody concentration of the half light absorption value under different antigen concentrations is calculated, and four antibody concentrations (mol/L) are obtained: 3.2576X 10^-8mol/L、3.2292×10^-8mol/L、2.0052×10^-8mol/L、2.4484×10^-8mol/L. The affinity constant was calculated by substituting the formula K ═ N-1)/(N × AB' -AB). AB 'and AB are antibody concentrations that produce half-absorbance values at the corresponding antigen concentrations AG (3mg/L, 1.5ml/L, 0.75mg/L, 0.375mg/L), N ═ AG/AG' (AG/AG)>AG'). When N is 2, three K values of 3.125, 12.8, 3.45 can be obtained. When N is 4, two K values 7.25, 4.57 can be obtained. When N is 8, a K value 4.287 is obtained. The average of 6K values (see Table 3) was found to be 5.9137X 10⁹It is demonstrated that the anti-human MVP monoclonal antibody provided in example 1 has a better affinity for MVP antigen.

TABLE 3

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications kyi may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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<400> 4

gacattgagc tcacccagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag 120

ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca 180

aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct 240

gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtggac gttcggtgga 300

ggcaccaagg tggaaattaa acgg 324

<210> 5

<211> 400

<212> PRT

<213> Artificial sequence

<400> 5

Met Ala Thr Glu Glu Phe Ile Ile Arg Ile Pro Pro Tyr His Tyr Ile

1 5 10 15

His Val Leu Asp Gln Asn Ser Asn Val Ser Arg Val Glu Val Gly Pro

20 25 30

Lys Thr Tyr Ile Arg Gln Asp Asn Glu Arg Val Leu Phe Ala Pro Met

35 40 45

Arg Met Val Thr Val Pro Pro Arg His Tyr Cys Thr Val Ala Asn Pro

50 55 60

Val Ser Arg Asp Ala Gln Gly Leu Val Leu Phe Asp Val Thr Gly Gln

65 70 75 80

Val Arg Leu Arg His Ala Asp Leu Glu Ile Arg Leu Ala Gln Asp Pro

85 90 95

Phe Pro Leu Tyr Pro Gly Glu Val Leu Glu Lys Asp Ile Thr Pro Leu

100 105 110

Gln Val Val Leu Pro Asn Thr Ala Leu His Leu Lys Ala Leu Leu Asp

115 120 125

Phe Glu Asp Lys Asp Gly Asp Lys Val Val Ala Gly Asp Glu Trp Leu

130 135 140

Phe Glu Gly Pro Gly Thr Tyr Ile Pro Arg Lys Glu Val Glu Val Val

145 150 155 160

Glu Ile Ile Gln Ala Thr Ile Ile Arg Gln Asn Gln Ala Leu Arg Leu

165 170 175

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

180 185 190

Gly Glu Glu Trp Leu Val Thr Thr Val Gly Ala Tyr Leu Pro Ala Val

195 200 205

Phe Glu Glu Val Leu Asp Leu Val Asp Ala Val Ile Leu Thr Glu Lys

210 215 220

Thr Ala Leu His Leu Arg Ala Arg Arg Asn Phe Arg Asp Phe Arg Gly

225 230 235 240

Val Ser Arg Arg Thr Gly Glu Glu Trp Leu Val Thr Val Gln Asp Thr

245 250 255

Glu Ala His Val Pro Asp Val His Glu Glu Val Leu Gly Val Val Pro

260 265 270

Ile Thr Thr Leu Gly Pro His Asn Tyr Cys Val Ile Leu Asp Pro Val

275 280 285

Gly Pro Asp Gly Lys Asn Gln Leu Gly Gln Lys Arg Val Val Lys Gly

290 295 300

Glu Lys Ser Phe Phe Leu Gln Pro Gly Glu Gln Leu Glu Gln Gly Ile

305 310 315 320

Gln Asp Val Tyr Val Leu Ser Glu Gln Gln Gly Leu Leu Leu Arg Ala

325 330 335

Leu Gln Pro Leu Glu Glu Gly Glu Asp Glu Glu Lys Val Ser His Gln

340 345 350

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

355 360 365

Ala Lys Val Glu Val Val Glu Glu Arg Gln Ala Ile Pro Leu Asp Glu

370 375 380

Asn Glu Gly Ile Tyr Val Gln Asp Val Lys Thr Gly Lys Val Arg Ala

385 390 395 400

Claims

1. An antibody of anti-human vault protein or an antigen binding fragment thereof is characterized in that the amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the antibody is shown as SEQ ID NO. 2.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is selected from any one of Fab ', Fab, F (ab')2, scFv, and Fv of the antibody.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the constant region of the antibody is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the species of origin of the constant region of the antibody is mouse, rat, bovine, horse, sheep, rabbit or dog.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the species of origin of the constant region of the antibody is mouse.

6. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 for the preparation of a diagnostic reagent or kit for tumors, wherein the diagnostic marker for tumors is human vault protein.

7. An immunoconjugate, bispecific molecule, chimeric antigen receptor or pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5.

8. A test kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

9. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-5.

10. A recombinant cell comprising a recombinant vector comprising the nucleic acid molecule of claim 9.

11. A method of producing the antibody or antigen-binding fragment thereof of any one of claims 1-5, comprising: culturing the recombinant cell of claim 10, and isolating and purifying the antibody or antigen-binding fragment thereof from the culture product.