CN114656559B

CN114656559B - Binding protein capable of specifically binding CK7 protein, kit and application thereof

Info

Publication number: CN114656559B
Application number: CN202210448113.2A
Authority: CN
Inventors: 付康; 赵逸堃
Original assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Current assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Priority date: 2022-04-26
Filing date: 2022-04-26
Publication date: 2023-06-02
Anticipated expiration: 2042-04-26
Also published as: CN114656559A

Abstract

The invention discloses a binding protein capable of specifically binding to CK7 protein, a kit and application thereof, and relates to the technical field of biology. The binding protein has better binding activity and affinity, the sensitivity and the specificity of detection can be improved by using the binding protein to detect CK7, the binding protein can be used for diagnosing diseases taking CK7 as a marker, and the binding protein provides more protein choices for detecting CK7 and diagnosing diseases taking CK7 as the marker.

Description

Binding protein capable of specifically binding CK7 protein, kit and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a binding protein capable of specifically binding to CK7 protein, a kit and application thereof.

Background

Cytokeratins (CKs) are the major skeletal proteins in epithelial cells or keratinocytes, and are structural proteins that maintain the integrity and continuity of epithelial tissue. At least 20 different keratin molecules in cytokeratin family are closely related to proliferation and differentiation of epithelial cells, are a common tumor immunohistochemical marker and are related to cancers derived from various epithelial cells. In patients with mesothelioma, etc., an increase in cytokeratin expression occurs. The detection of cytokeratin thus helps to assist in the diagnosis of malignancy.

CK7 protein, the positive site of which is cytoplasmic, is present in glandular and transitional epithelial cells of most normal tissues, and is not expressed by cells of generally non-epithelial origin. Ovarian serosity and positive response in endometrioid adenocarcinoma, breast carcinoma, lung adenocarcinoma, while adenocarcinoma of the gastrointestinal tract, ovarian mucinous adenocarcinoma, are negative. CK7 is now generally considered a relatively specific marker for adenocarcinoma and transitional epithelial cell carcinoma. The combined use with CK20 and Villin helps to identify the primary site of adenocarcinoma.

Clinical detection of protein expression in tumor cells is often performed by Immunohistochemical (IHC) pathology experiments, and the accuracy and sensitivity of the IHC experiments are determined by the quality of monoclonal antibodies that specifically bind the protein. Therefore, developing a monoclonal antibody against CK7 protein with higher binding specificity is of great importance for IHC detection of CK7 expression level.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a binding protein specifically binding to CK7 protein, a kit and application thereof, so as to solve the technical problems.

The binding protein provided by the invention can specifically bind to CK7, has better binding activity and affinity, and can improve the sensitivity and specificity of detection when being used for detecting CK7, and the binding protein can be used for diagnosing diseases taking PCT as a marker, such as tumors.

Noun definition

The term "binding protein" refers broadly to all proteins/protein fragments comprising CDR regions, in particular antibodies or antibody functional fragments. "antibody functional fragments" include antigen compound binding fragments of these antibodies, including Fab, F (ab') 2, fd, fv, scFv, bispecific antibodies, and antibody minimal recognition units, as well as single chain derivatives of these antibodies and fragments. The type of antibody may be selected from the group consisting of IgG1, igG2, igG3, igG4, and IgA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanzed) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".

The term "antibody" is used herein in its broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments so long as they exhibit the desired biological activity, such as specifically binding to an HRP-II antigen or fragment thereof. An "antibody fragment" includes a portion of a full length antibody, preferably an antigen binding or variable region thereof. Examples of antibody fragments include Fab, fab ', F (ab') 2, fd, fv, complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.

Typically, the variable regions VH/VL of the heavy and light chains of an antibody are obtained by joining the CDRs numbered below with the FR in a combination arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The invention is realized in the following way:

the present invention provides a binding protein that specifically binds to CK7 protein, comprising an antigen binding domain having 6 complementarity determining regions as shown below:

CDR-VH1：HYGVD；

CDR-VH2：VIWGIGSTDYNSVLMS；

CDR-VH3：RNYGSLEY；

CDR-VL1：RSSQSLVHRNGNTYLH；

CDR-VL2：KVSNRFS；

CDR-VL3：SQTTHVPPLT。

the amino acid sequence of the complementarity determining region was first discovered and revealed by the present invention, and is a novel sequence that confers upon the binding protein the ability to bind to the CK7 antigen. The binding protein has better binding activity and affinity, the sensitivity and the specificity of detection can be improved by using the binding protein for detecting CK7, and the binding protein can be used for diagnosing diseases taking CK7 as a marker.

In a preferred embodiment of the present invention, the binding protein and the CK7 protein are expressed as K _D ＝3.0907×10 ⁹ Is an antibody or a functional fragment.

In a preferred embodiment of the present invention, the binding protein comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, which are shown in sequence SEQ ID NO. 1-4, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, which are shown in sequence SEQ ID NO. 5-8.

The sequences of SEQ ID NOS 1-8 are shown in the following table:

in a preferred embodiment of the present invention, the binding protein is selected from any one of F (ab ') 2, fab', fab, fv, scFv and bispecific antibody.

The functional fragment of the above antibody generally has the same binding specificity as the antibody from which it is derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the antibodies described above may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds.

Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.

The invention also provides a hybridoma cell strain, which is named as 4C8.

The invention also provides application of the binding protein in preparing a reagent or a kit for diagnosing tumor or CK7 immunodetection.

In a preferred embodiment of the invention, the tumor is selected from the group consisting of epithelial tumors; CK7 immunodetection is to label tissue cells with binding proteins;

in an alternative embodiment, the epithelial tumor is selected from ovarian cancer, endometrial tumor, cervical cancer, lung cancer, adenocarcinoma, or adenoma;

in an alternative embodiment, the adenocarcinoma is selected from lung adenocarcinoma, breast cancer, thyroid cancer, salivary gland cancer, or pancreatic cancer;

adenomas are selected from cystic, fibroadenomas, polymorphic adenomas or polypoidal adenomas;

in an alternative embodiment, the breast cancer is selected from ductal breast cancer, epithelial breast cancer or lobular breast cancer;

in an alternative embodiment, the tissue cells are ovary, endometrium, cervix, lung, thyroid and mammary epithelium.

The invention also provides a reagent or a kit for diagnosing tumor or CK7 immunoassay, which contains the binding protein;

in an alternative embodiment, the tumor is selected from the group consisting of epithelial tumors;

in an alternative embodiment, the epithelial tumor is selected from adenocarcinoma, lung carcinoma or ovarian mucinous carcinoma.

In an alternative embodiment, the adenocarcinoma is selected from lung adenocarcinoma, breast cancer or pancreatic cancer;

in an alternative embodiment, the breast cancer is selected from ductal breast cancer or lobular breast cancer.

The invention also provides a vector comprising a nucleic acid encoding the binding protein described above.

The invention also provides a host cell which contains the vector.

Also provided is a method of producing the binding protein of any one of the preceding embodiments, comprising:

culturing the host cell of the previous embodiment, and isolating and purifying the binding protein from the culture medium or from the cultured host cell.

The production method may be, for example, transfection of a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions to express the binding protein. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the binding protein. The binding proteins may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.

Construction of a suitable vector containing the coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailing and religated as desired. Mutations may be introduced into the coding sequence by any method to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions, etc.

A method of detecting CK7, comprising: mixing the binding protein of any of the preceding embodiments with a test sample.

In alternative embodiments, the above-described methods are for the purpose of diagnosis of non-disease.

It should be noted that, a person skilled in the art can perform qualitative or quantitative detection of CK7 protein in a sample to be detected based on the characteristics of antibody/antigen binding to form an immune complex.

The invention has the following beneficial effects:

the hybridoma cell strain and the binding protein produced by the hybridoma cell strain can be used for preparing a tumor diagnosis kit, and a diagnosis marker is Cytokeratin7 (CK 7, cytokeratin 7), so that the expression level of CK7 in relevant tumor tissues is detected.

Drawings

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

FIG. 1 is a graph showing the results of detection of endogenous CK7 proteins in a HeLa cell line (HeLa cells) when cell supernatants of cell lines 4C6 were subjected to Immunocytochemistry (ICC) detection;

fig. 2 is: immunohistochemical staining of lung adenocarcinoma slice tissues;

fig. 3 is: immunohistochemical experimental results;

fig. 4 is: recombinant human CK7 protein sequence.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

This example demonstrates the preparation of monoclonal antibodies specific for CK7 protein.

1 immunization of animals (mice)

Mice were immunized with the immunogen using a universal method. The immunogen is recombinant human CK7 protein with His tag, and is used as detection antigen to measure and screen serum titer and hybridoma, and the high-purity antigen can increase the opportunity of obtaining the required monoclonal antibody and reduce the screening amount. 5 mice were immunized, each immunized 50ug CK7 antigen. The antigen protein solution was prepared with PBS. And (3) placing a proper amount of antigen protein, PBS and Freund's adjuvant into a syringe, plugging a water outlet of the syringe by a plug, and placing the syringe on an emulsifying instrument for stirring and fully emulsifying to ensure that the antigen adjuvant forms a stable water-in-oil solution. The first tail blood serum titer detection is carried out 7-10 days after primary and secondary immunity, and good titer is obtained after 2-4 times of booster immunization. Cell fusion is carried out after the immune mice with high serum titer are selected for abdominal cavity termination.

The recombinant human CK7 protein sequence is shown with reference to fig. 4.

2 hybridoma cell fusion and selection

Readiness is required prior to cell fusion: 1. culturing mouse myeloma cells SP2/0 to log phase of growth; 2. one negative mouse was sacrificed the day before fusion, and mouse peritoneal trophoblast cells were taken from the mouse peritoneal cavity with HAT medium injected into the mouse peritoneal cavity in a sterile environment and plated in 96-well plates, 100ul per well, with these cells promoting hybridoma cell growth. Immunized mice were sacrificed, spleens were removed under sterile conditions, splenocytes and SP2/0 myeloma cells were chemically fused with PEG, appropriate HAT medium was added according to the number of plates to be plated, and finally the fused cells were plated on trophoblast cell culture plates at 100ul per well.

The growth of surviving hybridoma cells was observed under a microscope after 7-10 days, and after two weeks of plating, the supernatants of each well were collected and hybridoma cells were screened by ELISA using human CK7-his protein antigen. The method comprises the following steps: the ELISA plate was coated with 100ul 2ug/ml of human CK7-his protein antigen in PBS for two hours at 37 ℃. After 3 PBST washes, 150 ul/well of 3% nonfat dry milk in PBS was added and blocked overnight at 4 ℃. Plates were washed three more times, 80 ul/well hybridoma supernatant was added, incubated for 1 hour at 37℃and plates were washed three more times. Add 1 at 100 ul/well: and 7000 dilution of horseradish peroxidase-labeled goat anti-mouse secondary antibody, incubation at 37 ℃ for 45 minutes, plate washing for 3 times and drying. 100 ul/well TMB color development was added, developed for 5-10 minutes at room temperature, stopped with 2M sulfuric acid solution, and absorbance was measured for each well at 450 nm. Positive hybridoma cells were selected.

ELISA positive fusion wells were selected and then subjected to immunocytochemistry staining (ICC), and positive wells were selected for subsequent experiments. The experimental procedure was as follows: (1) After the adherent cells Hela cells are digested by pancreatin digestive juice in a few days before the experiment, the cells are paved in a 96-well plate, and the adherent cells are fully paved with the well plate; (2) taking out the 96-well plate, pouring out the culture medium, and washing 3 times with PBS; (3) Adding 4% paraformaldehyde, fixing at room temperature for 15-30 min, and washing the plate with PBS for three times; (4) 0.2% triton X-100 room temperature for 10-15min, PBS rinse three times; (5) 3% hydrogen peroxide for 15min, and PBS rinsing for 3 times; 6.3% goat serum was blocked for 1 hour at 37 ℃; (6) 80ul of the fusion well ELISA positive cell supernatant was added to the icc assay plate overnight at 4 ℃; (7) after PBST rinsing, adding secondary antibody to react for 40min at room temperature; (8) DAB color development; (9) hematoxylin staining; (10) PBST anti-blue observations. The experimental results are shown with reference to fig. 1.

Fusion cells which are positive in combination are selected through ELISA and ICC experiments, cloned through a limiting dilution method, and each positive strain is paved into a 48/96-well plate for continuous culture. And performing secondary screening by ELISA method, screening out hybridoma which specifically recognizes CK7 protein and can block CK7 binding, subcloning by limiting dilution method, and obtaining a monoclonal cell strain 4c6. Expanding the monoclonal cell line to collect about 1×10 ⁶ Individual cells are injected into selected mice (the mice need to be injected with paraffin oil in the abdominal cavity in advance for one week), and after waiting for 7-10 days, the mice generate ascites, and the ascites is collected for antibody purification. After purification, a mouse monoclonal antibody against the CK7 protein was obtained.

Example 2

DNA cloning and sequencing was performed and the variable region gene of the anti-human CK7 monoclonal antibody was sequenced.

Total RNA was extracted from the mouse monoclonal cell line using Trizol reagent. Collect in 9cm dishThe cultured cells were placed in a 1.5ml centrifuge tube, and the supernatant was aspirated to dryness. 1ml of Trizol reagent was added and the cells were blown up uniformly to lyse the cells, and the lysed sample or homogenate was left at room temperature for 5-10min to allow complete separation of nucleoprotein from nucleic acid. 0.2ml of chloroform was added thereto, and the mixture was vigorously shaken for 15sec and left at room temperature for 3min. Centrifuge at 12000rpm at 4℃for 10min. The upper aqueous phase is sucked and transferred into a clean centrifuge tube, added with isopropyl alcohol with equal volume, evenly mixed and placed for 20min at room temperature. Centrifuge at 12000rpm at 4℃for 10min, discard supernatant. The precipitate was washed with 1ml of 75% ethanol. Centrifuge at 12000rpm at 4℃for 3min, discard supernatant. Drying at room temperature for 5-10min. Adding 30-50ul RNase-free ddH ₂ O. The resulting RNA solution was stored at-70℃or used in subsequent experiments.

The total RNA is reverse transcribed into cDNA by using AMV first-strand cDNA synthesis kit. The experimental system was configured as follows, with 6ul total RNA+1ul Oligo dT+4ul RNase-free water (11 ul total). After gentle mixing, the mixture was centrifuged for 3-5s, and after 5mi of pre-denaturation in a warm bath at 65℃the reaction mixture was ice-cooled for 30s, and then centrifuged for 3-5s followed by 2min in an ice-cooled bath. Under ice bath, 4ul of 5 Xbuffer+1 ul of dNTP mixture+1 ul of RNase inhibitor+1 ul of reverse transcriptase (total 20ul system) were added, and after gentle mixing, the mixture was centrifuged for 3-5s, and on a PCR instrument, the mixture was subjected to 42℃for 50 minutes and 85℃for 5 minutes to complete cDNA synthesis. Random primers are suitable for the synthesis of short-chain cDNA below 500bp, and the transcribed RNA template can transcribe 5' -terminal regions without poly (A) tail.

PCR amplification of light and heavy chains. For amplifying antibody light chain variable region sequences, a PCR reaction system was configured: 2 XTaq enzyme buffer 25ul+FP-VL 1ul+RP-VL 1ul+cDNA 2ul+ddH ₂ And O21 ul. For amplifying antibody heavy chain variable region sequences, a PCR reaction system was configured: 2 XTaq enzyme buffer 25ul+FP-VH1ul+RP-VH1ul+cDNA 2ul+ddH ₂ And O21 ul. The temperature cycle for PCR amplification of the heavy and light chain variable regions is as follows (wherein steps 2 to 4, 35 cycles are repeated):

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

step 2-denaturation 94℃for 30sec;

step 3-annealing at 55 ℃ for 45sec;

step 4-extending at 72 ℃ for 60sec;

step 5-72 ℃ for 10min;

step 6-storing at 4 ℃.

The PCR products were analyzed by 1% agarose gel electrophoresis, bands of DNA segments of the corresponding sizes (approximately 325bp for VH and approximately 325bp for VL) were excised and DNA extracted using the SanPrep column DNA gel recovery kit. The following is a brief description: cutting off a gel block containing the target fragment from agarose gel, and weighing; adding buffer B2 with the weight 3-6 times of that of the rubber block, and carrying out water bath at 50 ℃ for 5-10 minutes to obtain sol; transferring the sol into an adsorption column, and centrifuging 8000g for 30 seconds; pouring out the liquid in the collecting pipe; adding 500ul wash Solution,9000g into the column, centrifuging for 30 seconds, and pouring out the liquid in the collecting pipe; repeatedly adding the wash solution once, and pouring out the liquid; centrifuging the hole adsorption column at 9000g for 1 min; the column was placed in a clean 1.5ml centrifuge tube, 15-40 g ul Elution Buffer g was added to the center of the adsorption membrane, and after 1 min of standing at room temperature, it was centrifuged for 1 min. The prepared DNA solution is obtained, and the variable region sequence of the antibody is obtained by purifying the PCR product and sequencing.

Experimental example 1

This experimental example detects the affinity and sensitivity of the antibody prepared in example 1.

1. Antigen was plated at 3mg/L, 1.5mg/L, 0.75mg/L, 0.375 mg/L.

2. Adjusting the antibody concentration to 10 ^-7 mol/L level (1X 10 ^-7 To 5x 10 ^-7 mol/L). Then diluted 1:2-1:256 in a multiple ratio and added to wells with different antigen coating amounts.

3. Adding secondary antibody and developing TMB. The absorbance at 450nm was measured and the data is shown in Table 1.

4. And (3) according to the antigen-antibody combination S curve, determining the antibody concentration of half-absorbance values under different antigen concentrations. Thus there are four antibody concentrations (mol/L). 4.615*10 ^-8 mol/L、3.576*10 ^-8 mol/L、3.422*10 ^-8 mol/L、3.622*10 ^-8 mol/L。

5. The affinity constant is calculated by substituting the formula k= (N-1)/(n×ab' -AB). AB', AB is the concentration of antibody that gave half-absorbance at the corresponding antigen concentrations AG (3 mg/L, 1.5ml/L, 0.75mg/L, 0.375 mg/L). n=ag/AG '(AG > AG').

6. When n=2When three K values 3.9417, 3.0600, 2.6151 are available. When n=4, two K values 3.3065, 2.7483 can be obtained. When n=8, a K value 2.8725 is obtained, an average of 6K values is obtained of 3.0907×10 ⁹ 。

TABLE 1

Experimental example 2

The binding capacity of purified antibody CK7-4C8 to the CK7 protein was determined based on Immunohistochemistry (IHC).

Baking lung adenocarcinoma slices in a 60 ℃ incubator for 60 minutes, soaking the slices in xylene I for 15 minutes, replacing xylene II, and then soaking for 15 minutes, wherein the slices are respectively soaked in absolute ethyl alcohol (1) for 5 minutes, absolute ethyl alcohol (2) for 5 minutes, 95% ethyl alcohol for 5 minutes, 85% ethyl alcohol for 5 minutes and 75% ethyl alcohol for 5 minutes; soaking ddH2O for 5 minutes, and cleaning for 3 times; adding 10mmol/L citrate buffer solution (ph 6.0) which is enough to submerge slices into a pressure cooker for antigen retrieval (boiling method), heating to boiling, placing the slices on a hot material slice frame, placing into the cooker, covering a cooker cover, fastening a pressure valve, continuing heating, setting pressure maintaining for 4 minutes, opening a deflation valve for deflation after the time is up, opening the cooker cover after the pressure is zeroed, taking out the inner cooker, and cooling at room temperature. Taking out the slice (about 40 min) after the solution is cooled to room temperature; ddH2O is soaked for 5 minutes, washed for 2 times, PBST is soaked for 5 minutes, and washed for 2 times; the sections were placed in 20ml of 3% H2O 2-methanol solution and treated at room temperature for 10 minutes in the absence of light; PBST is soaked for 5 minutes and is washed for 3 times; one drop of goat serum blocking solution was added to each tissue group and incubated in a wet box for 45 minutes at room temperature; PBST was soaked for 5 minutes and washed 3 times. The treated tissue sections were compared with CK7 antibody from Roche, and the remaining sections were subjected to CK7-4C8 purification. Incubation overnight in a wet box at 4 ℃; taking out from the refrigerator at the temperature of 4 ℃ and incubating for 60 minutes at room temperature; PBST is gently washed and then soaked for 5 minutes, and the PBST is washed for 3 times; each tissue group was incubated with HRP-labeled Changdao secondary antibody (CAT#:) 25ul at room temperature for 45 min; washing; preparing DAB color development liquid, reacting for 10-15min in dark, then dripping the DAB color development liquid onto a slice, and developing for 1-5 min; terminating the color reaction with distilled water; dropping 50ul hematoxylin dye solution into each tissue group, dyeing for 5-10 minutes, and washing with distilled water; the slices are put into 1 percent hydrochloric acid-ethanol for decoloration for 2 to 3 seconds, then are quickly taken out and put into distilled water for termination, and then are put into PBST (pH 8.0) for blue reflection for 5 to 10 minutes; soaking in 75% ethanol for 5 min: soaking in 85% ethanol for 5 min; soaking in 95% ethanol for 5 min: soaking in absolute ethanol for 5 minutes. Soaking in xylene for 10min, and soaking for 10min after changing xylene; dripping a neutral resin sealing piece, and covering a glass slide sealing piece; microscope imaging. As shown in fig. 2 and 3.

Immunohistochemical neutralization assay: a certain amount of CK7 antigen protein is taken and mixed with CK7-4C8 purified antibody, and neutralization reaction is carried out at 37 ℃ for 1 hour. The method comprises the steps of performing immunohistochemical experiments, dripping the antibody serving as a primary antibody to lung adenocarcinoma slices, overnight at 4 ℃, performing immunohistochemical staining according to the same steps, and taking a picture.

FIGS. 2 and 3 show immunohistochemical experiments of CK7, and lung adenocarcinoma tissues of different patients are detected as samples. FIG. 2A shows the CK7-4C8 antibody produced by us, and FIG. 2B shows the CK7 antibody from Roche. FIG. 3A shows the CK7-4C8 antibody produced by us, and FIG. 3B shows the CK7 antibody from Roche. As can be seen from the staining results of FIGS. 2 and 3, the CK7-4C8 antibody and the Roche antibody have no obvious difference in staining sites and staining intensity. The CK7 provided by the invention has application prospect in preparing a kit.

The neutralization test results are shown as C in FIG. 3. The antigen protein reaction in the neutralization assay binds to the CK7-4C8 antibody, which cannot bind to the antigen in the cancer tissue, leaving the tissue almost stained. The anti-CK 7 protein monoclonal antibody CK7-4C8 provided by the embodiment of the invention can recognize and combine with CK7 protein in human cells, so that cytoplasms in lung adenocarcinoma tissues are brown in subsequent IHC staining experiments, and no obvious difference is caused between the anti-CK 7 protein monoclonal antibody CK7-4C8 and CK7 antibodies of Roche company.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. 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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<120> a binding protein specifically binding to CK7 protein, kit and use thereof

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Claims

1. A binding protein that specifically binds CK7 protein, comprising an antigen binding domain having 6 complementarity determining regions as set forth in:

CDR-VH1：HYGVD；

CDR-VH2：VIWGIGSTDYNSVLMS；

CDR-VH3：RNYGSLEY；

CDR-VL1：RSSQSLVHRNGNTYLH；

CDR-VL2：KVSNRFS；

CDR-VL3：SQTTHVPPLT。

2. the binding protein that specifically binds to CK7 protein according to claim 1, wherein the binding protein binds to CK7 protein in K _D ≤3.0907×10 ⁹ Is an antibody or a functional fragment.

3. The binding protein for specifically binding to CK7 protein according to any one of claims 1 to 2, characterized in that it comprises the light chain framework regions FR1-L, FR-L, FR3-L and FR4-L and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-L and respectively having the sequences shown in SEQ ID NO. 1-4 and SEQ ID NO. 5-8, respectively.

4. The binding protein that specifically binds to CK7 protein according to any one of claims 1-2, wherein the binding protein is selected from any one of F (ab ') 2, fab', fab, fv, scFv, and bispecific antibodies.

5. Use of a binding protein according to any one of claims 1 to 4 for the preparation of a reagent or kit for diagnosing an epithelial tumor or CK7 immunodetection.

6. The use according to claim 5, wherein said CK7 immunoassay is labelling of tissue cells with said binding protein.

7. The use according to claim 6, wherein the tissue cells are ovary, endometrium, cervix, lung, thyroid and mammary gland epithelium.

8. The use according to claim 5, wherein the epithelial tumor is selected from ovarian cancer, endometrial tumor, cervical cancer, lung cancer, adenocarcinoma or adenoma.

9. The use according to claim 8, wherein the adenocarcinoma is selected from lung adenocarcinoma, breast carcinoma, thyroid carcinoma, salivary gland carcinoma or pancreatic carcinoma;

the adenoma is selected from the group consisting of cystic, fibroadenoma, polymorphic adenoma and polypoidal adenoma.

10. The use according to claim 9, wherein the breast cancer is selected from ductal breast cancer, epithelial breast cancer or lobular breast cancer.

11. A reagent or kit for diagnosing an epithelial tumor or a CK7 immunoassay, comprising the binding protein of any one of claims 1-4.

12. The reagent or kit for diagnosing an epithelial tumor or CK7 immunodetection according to claim 11, wherein the epithelial tumor is selected from the group consisting of adenocarcinoma and lung cancer.

13. The reagent or kit for diagnosing epithelial tumors or CK7 immunological tests according to claim 12, wherein the adenocarcinoma is selected from lung adenocarcinoma, ovarian mucinous carcinoma, breast cancer or pancreatic cancer.

14. The reagent or kit for diagnosing epithelial tumors or CK7 immunodetection of claim 13, wherein the breast cancer is selected from the group consisting of ductal breast cancer and lobular breast cancer.

15. A vector comprising a nucleic acid encoding the binding protein of any one of claims 1-4.

16. A host cell comprising the vector of claim 15.