CN114656559A

CN114656559A - Binding protein specifically binding CK7 protein, kit and application thereof

Info

Publication number: CN114656559A
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: 2022-06-24
Anticipated expiration: 2042-04-26
Also published as: CN114656559B

Abstract

The invention discloses a binding protein specifically binding 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 detection sensitivity and specificity can be improved by using the binding protein to detect CK7, the binding protein can be used for diagnosing diseases with CK7 as a marker, and the invention provides more protein choices for the detection of CK7 and the diagnosis of diseases with CK7 as a marker.

Description

Binding protein specifically binding CK7 protein, kit and application thereof

Technical Field

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

Background

Cytokeratins (CKs) are the major skeletal proteins in epithelial or keratinocyte cells, as well as structural proteins that maintain the integrity and continuity of epithelial tissue. The cytokeratin family has at least 20 different keratin molecules, is closely related to the proliferation and differentiation of epithelial cells, is a common tumor immunohistochemical marker and is related to cancers of various epithelial cell sources. In patients with mesothelioma or the like, cytokeratin expression is increased. The detection of cytokeratins is therefore useful to aid in the diagnosis of malignancies.

The CK7 protein, positive site is cytoplasm, is present in glandular epithelium and transitional epithelium cells of most normal tissues, and is not expressed by cells of non-epithelial origin generally. The ovarian serous and endometrioid adenocarcinoma, breast cancer and lung adenocarcinoma show positive reaction, while the adenocarcinoma of the gastrointestinal tract and the ovarian mucinous adenocarcinoma are negative. CK7 is now generally recognized as a relatively specific marker of adenocarcinoma and transitional epithelial carcinoma. The combined use with CK20 and Villin helps to determine the primary site of adenocarcinoma.

Clinically, the status of protein expression in tumor cells is usually detected by Immunohistochemical (IHC) pathological experiments, and the accuracy and sensitivity of IHC detection are determined by the quality of monoclonal antibodies specifically binding to proteins. Therefore, the development of a monoclonal antibody aiming at the CK7 protein with higher binding specificity has important significance for detecting the CK7 expression level by IHC.

In view of the above, the present invention is particularly proposed.

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 be specifically bound with CK7, has better binding activity and affinity, can improve the sensitivity and specificity of detection when being used for detecting CK7, and can be used for diagnosing diseases taking PCT as a marker, such as tumors.

Noun definitions

The term "binding protein" broadly refers to all proteins/protein fragments, in particular antibodies or functional fragments of antibodies, comprising CDR regions. "antibody functional fragments" include antigenic compound-binding fragments of such antibodies as described above, including Fab, F (ab')2, Fd, Fv, scFv, diabodies, and minimum recognition units of antibodies, as well as single chain derivatives of such antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, 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 (humanized) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".

The term "antibody" is used herein in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments so long as they exhibit the desired biological activity, e.g., specifically bind to an HRP-II antigen or fragment thereof. "antibody fragments" include portions of full-length antibodies, preferably the antigen-binding or variable regions 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.

In general, the variable domains VH/VL of the heavy and light chains of an antibody can be obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

The invention is realized by the following steps:

the invention provides a binding protein specifically binding to CK7 protein, the binding protein comprises an antigen binding structural domain, the antigen binding structural domain has 6 complementarity determining regions shown as follows:

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 is found and revealed for the first time in the invention, and is a novel sequence that can confer the binding protein the ability to bind to the CK7 antigen. The binding protein has good binding activity and affinity, the binding protein can be used for detecting CK7, the detection sensitivity and specificity can be improved, the binding protein can be used for diagnosing diseases with CK7 as a marker, and the invention provides more protein choices for detecting CK7 and diagnosing diseases with CK7 as a marker.

In a preferred embodiment of the invention, the binding protein and CK7 protein are expressed as K_D＝3.0907×10⁹The binding protein is an antibody or a functional fragment.

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

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

in a preferred embodiment of the invention, the binding protein is selected from the group consisting of F (ab ')2, Fab', Fab, Fv, scFv and diabody.

Functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. As will be readily understood by those skilled in the art based on the teachings of the present invention, functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to cleave disulfide bonds.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized 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 4C 8.

The invention also provides application of the binding protein in preparing a reagent or a kit for diagnosing tumors or performing CK7 immunoassay.

In a preferred embodiment of the invention, said tumor is selected from the group consisting of epithelial tumors; CK7 immunoassay is the labeling of tissue cells with binding proteins;

in an alternative embodiment, the epithelial tumors are selected from ovarian cancer, endometrioid tumors, 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;

adenoma is selected from the group consisting of cystadenoma, fibroadenoma, pleomorphic adenoma, or polypoid adenoma;

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 cell is ovarian, endometrial, cervical, lung, thyroid, and mammary epithelium.

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

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

in an alternative embodiment, the epithelial tumors are selected from adenocarcinoma, lung cancer or ovarian mucinous carcinoma.

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

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

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

The invention also provides a host cell containing the vector.

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

the host cell of the previous embodiment is cultured, and the binding protein is isolated and purified from the culture medium or from the cultured host cell.

The production method may be, for example, transfecting 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 such that the binding protein is expressed. 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 bound protein 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 suitable vectors containing the coding and regulatory sequences of interest can be carried out using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailed and religated as desired. Any method may be used to introduce mutations into the coding sequence to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions or the like.

A method of detecting CK7, comprising: mixing a binding protein according to any one of the preceding embodiments with a sample to be tested.

In an alternative embodiment, the above method is for the purpose of non-disease diagnosis.

It should be noted that, one skilled in the art can perform qualitative or quantitative detection of CK7 protein in the sample to be tested based on the characteristics of the immune complex formed by the antibody/antigen combination.

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 Cytokeratin 7(CK7, cytokine 7), so that the expression level of CK7 in related tumor tissues is detected.

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 graph showing the results of an Immunocytochemistry (ICC) assay of cell supernatant of cell line 4C6 on the recognition of endogenous CK7 protein in HeLa cell line (Hela cells);

FIG. 2 is a diagram of: immunohistochemically staining lung adenocarcinoma section tissues;

FIG. 3 is a diagram of: immunohistochemical experimental results;

FIG. 4 is a diagram of: recombinant human CK7 protein sequence.

Detailed Description

Reference will now 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. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).

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

This example was carried out to prepare a monoclonal antibody specific against CK7 protein.

1 animal (mouse) immunization

Mice are immunized with the immunogen using a general method. The immunogen is recombinant human CK7 protein with His label, and is used as detection antigen to measure and screen serum titer and hybridoma, and the high purity antigen can increase the chance of obtaining the required monoclonal antibody and reduce the screening amount. 5 mice were immunized, each mouse immunized with 50ug CK7 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 sequence of the recombinant human CK7 protein is shown in a figure 4.

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. one negative mouse was sacrificed one day before the fusion, and mouse peritoneal trophoblast cells were taken by injecting the mouse peritoneal cavity with HAT medium in a sterile environment and plated in 96-well plates at 100ul per well, and these cells had an effect of promoting the growth of hybridoma cells. Immunized mice were sacrificed, spleens were removed under sterile conditions, splenic B cells and SP2/0 myeloma cells were chemically fused with PEG, appropriate HAT medium was added according to the number to be plated, and finally the fused cells were plated on trophoblast cell culture plates at 100ul per well.

After 7-10 days, the growth of viable hybridoma cells can be observed under a microscope, and after two weeks of plating, supernatants from each well are collected and screened for hybridoma cells using the human CK7-his protein antigen by ELISA. The method comprises the following steps: the ELISA plates were coated with 100ul of 2ug/ml human CK7-his protein antigen in PBS for two hours at 37 ℃. After PBST washing 3 times, add 150 ul/well 3% skim milk powder in PBS and block at 4 ℃ overnight. The plate was washed three more times, 80 ul/well 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. 100 ul/well of TMB developing solution was added, development was carried out at room temperature for 5 to 10 minutes, 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 Hela cell 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 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, room temperature 10-15min, PBS rinse three times; (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) 80ul of cell supernatant of fusion well ELISA positive cells is added to an icc detection plate for 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 inverted blue observation. The experimental results are shown in FIG. 1.

Positive binding was picked by ELISA and ICC experimentsThe fused cells of (2) were cloned by limiting dilution method, and each positive strain was plated on 48/96 well plates and cultured. And performing a second round of re-screening by an ELISA method, screening out a hybridoma which specifically recognizes CK7 protein and can block the combination of CK7, and performing subcloning by a limiting dilution method to obtain a monoclonal cell strain 4c 6. 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 specific against CK7 protein was obtained.

Example 2

DNA cloning and sequencing were performed, and variable region genes of the anti-human CK7 monoclonal antibody were sequenced.

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 12000rpm for 10min at 4 ℃. Absorbing the upper layer water phase, transferring 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 30-50ul 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 set-up was as follows, 6ul total RNA +1ul Oligo dT +4ul RNase-free water (total 11 ul). 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. 4ul of 5 Xbuffer solution +1ul of dNTP mixture +1ul of RNase inhibitor +1ul of reverse transcriptase (20 ul system in total) were added under ice bath conditions, gently mixed, centrifuged for 3-5s, and subjected to PCR at 42 ℃ for 50 minutes and 85 ℃ for 5 minutes to complete 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 25ul + FP-VL 1ul + RP-VL 1ul + cDNA 2ul + ddH₂O21 ul. For the amplification of the heavy chain variable region sequence of the antibody, a PCR reaction system is configured: 2 xTaq enzyme buffer 25ul + FP-VH 1ul + RP-VH 1ul + cDNA 2ul + ddH₂O21 ul. 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 product was analyzed by 1% agarose gel electrophoresis, and a band of DNA segment of a corresponding size (about 375bp for VH and about 325bp for VL) was excised, and DNA extraction was performed using a DNA gel recovery kit of 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; 500ul wash Solution is added into the column, and the column is centrifuged for 30 seconds at 9000g, and the liquid in the collecting pipe is poured out; adding wash solution repeatedly once again to pour 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, 15-40ul of Elution Buffer was added to the center of the adsorption membrane, and after standing at room temperature for 1 minute, centrifugation was carried out for 1 minute. Obtaining the prepared DNA solution, purifying PCR products and obtaining the variable region sequence of the antibody through sequencing.

Experimental example 1

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

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

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

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

4. And (4) according to the antigen-antibody combination S curve, calculating the antibody concentration of the half light absorption value under different antigen concentrations. Thus, there were four antibody concentrations (mol/L). 4.615*10^-8mol/L、3.576*10^-8mol/L、3.422*10^-8mol/L、3.622*10^-8mol/L。

5. The affinity constants were calculated by substituting the formula K ═ (N-1)/(N × AB' -AB). AB', AB are the antibody concentrations that give half the absorbance values at the corresponding antigen concentrations AG (3mg/L, 1.5ml/L, 0.75mg/L, 0.375 mg/L). N ═ AG/AG '(AG > AG').

6. When N is 2, three K values 3.9417, 3.0600, 2.6151 can be obtained. When N is 4, two K values 3.3065, 2.7483 can be obtained. When N is 8, a K value 2.8725 is obtained, and an average value of 3.0907 x 10 is obtained by averaging 6K values⁹。

TABLE 1

Experimental example 2

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

Placing lung adenocarcinoma slices in a constant temperature box at 60 ℃ for baking for 60 minutes, soaking the slices in xylene I for 15 minutes, replacing the xylene II, soaking for 15 minutes, and respectively soaking in absolute ethyl alcohol I for 5 minutes, absolute ethyl alcohol II 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 washing for 3 times; adding 10mmol/L citrate buffer solution (ph6.0) into a pressure cooker for antigen retrieval (boiling method) by using the pressure cooker, heating to boil, placing slices on a heat-resistant material slice rack, placing the slices into the pressure cooker, covering the pressure cooker cover, fastening a pressure valve, continuing heating, maintaining the pressure for 4 minutes, opening an air release valve to release air after the time is up, opening the pressure cooker cover after the pressure returns to zero, taking out the inner cooker, and cooling at room temperature. The sections were removed after the solution was cooled to room temperature (about 40 min); ddH2O is soaked for 5 minutes and washed for 2 times, PBST is soaked for 5 minutes and washed for 2 times; the slices were placed in 20ml of 3% H2O 2-methanol solution, protected from light and treated 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 amount of the goat serum blocking solution is about 25ul, and incubating for 45 minutes at room temperature in a wet box; PBST was soaked for 5 minutes and washed 3 times. Treated tissue sections were compared with CK7 antibody from Roche, and the remaining sections were purified by addition of CK7-4C 8. Incubation in a wet box at 4 ℃ overnight; 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; 25ul of HRP-labeled Long island secondary antibody (CAT #: 25 u) was added to each tissue group, and 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 50ul of hematoxylin staining solution into each tissue group, staining for 5-10 minutes, 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 and sealing, and covering with glass slide; and (5) shooting with a microscope. As shown in fig. 2 and 3.

Immunohistochemical neutralization experiments: a certain amount of CK7 antigen protein is mixed with CK7-4C8 purified antibody, and neutralization reaction is carried out for 1 hour at 37 ℃. The cells were subjected to immunohistochemical experimental procedures, as a single anti-drop, to lung adenocarcinoma sections at 4 ℃ overnight, followed by immunohistochemical staining and photography according to the same procedures.

Fig. 2 and fig. 3 show immunohistochemical test of CK7, and the test specimens are lung adenocarcinoma tissues of different patients. In FIG. 2, A is CK7-4C8 antibody produced by us, and B is CK7 antibody of Roche. In FIG. 3, A is CK7-4C8 antibody produced by us, and B in FIG. 3 is CK7 antibody of Rogowski company. As is clear from the staining results in FIGS. 2 and 3, the CK7-4C8 antibody and Roche antibody showed no significant difference in staining site and staining intensity. The CK7 provided by the invention is proved to have an application prospect in preparation of the kit.

In fig. 3, C is the result of the neutralization experiment. The antigen protein response in the neutralization assay bound the CK7-4C8 antibody, which failed to bind to the antigen in the cancer tissue, resulting in little staining of the tissue. It can be seen that the anti-CK 7 protein monoclonal antibody CK7-4C8 provided by the embodiment of the invention can recognize and bind to CK7 protein in human cells, and enables cell cytoplasm in lung adenocarcinoma tissues to be brown in subsequent IHC staining experiments, and the cell cytoplasm is not obviously different from CK7 antibody of Roche.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes 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

<110> Biotechnology engineering (Shanghai) Ltd

<120> binding protein specifically binding CK7 protein, kit and application thereof

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<170> PatentIn version 3.5

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

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

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

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Phe Gly Ala Gly Thr Lys Pro Glu Ile Lys

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

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Claims

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

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 with K_D≤3.0907×10⁹The binding protein is an antibody or functional fragment.

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

4. The binding protein that specifically binds to the CK7 protein according to any one of claims 1 to 3, wherein the binding protein is selected from any one of F (ab ')2, Fab', Fab, Fv, scFv, and diabody.

5. Use of a binding protein according to any one of claims 1 to 4 in the preparation of a reagent or kit for the diagnosis of a tumor or an immunoassay for CK 7.

6. The use according to claim 5, wherein said tumor is selected from the group consisting of epithelial tumors; the CK7 immunoassay is used for labeling tissue cells by the binding protein;

preferably, the epithelial tumors are selected from ovarian cancer, endometrioid tumors, cervical cancer, lung cancer, adenocarcinoma or adenoma;

preferably, the adenocarcinoma is selected from lung adenocarcinoma, breast cancer, thyroid cancer, salivary gland cancer or pancreatic cancer;

said adenoma is selected from the group consisting of cystadenoma, fibroadenoma, pleomorphic adenoma, or polypoid adenoma;

preferably, the breast cancer is selected from ductal carcinoma of the breast, epithelial carcinoma of the breast or lobular carcinoma of the breast;

preferably, the tissue cells are ovarian, endometrial, cervical, lung, thyroid and mammary epithelium.

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

preferably, the tumor is selected from epithelial tumors;

preferably, the epithelial tumors are selected from adenocarcinoma, lung cancer or ovarian mucinous carcinoma;

preferably, the adenocarcinoma is selected from lung adenocarcinoma, breast cancer or pancreatic cancer.

8. The reagent or the kit for diagnosing tumor or CK7 immunodetection according to claim 7, wherein the breast cancer is selected from ductal carcinoma of breast or lobular carcinoma of breast.

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

10. A host cell comprising the vector of claim 9.