CN112940118B

CN112940118B - Monoclonal antibody of anti-CK 8 protein, cell strain, preparation method and application thereof

Info

Publication number: CN112940118B
Application number: CN202110383269.2A
Authority: CN
Inventors: 蒋宁城; 杨清海; 陈惠玲; 王小亚; 傅椿辉; 林婷
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2022-04-12
Anticipated expiration: 2041-04-09
Also published as: CN112940118A

Abstract

The invention relates to a monoclonal antibody capable of identifying a human leukocyte differentiation antigen CK8, a secretory cell strain, a preparation method thereof and application thereof in immunodetection. The technical scheme includes that an amino acid sequence from 340 to 365 at the C terminal of CK8 protein is selected as an antigen peptide, and the antigen peptide is coupled with KLH carrier protein to obtain the immunogen; the amino acid sequence of the antigen peptide is shown as SEQ ID1, the mouse is immunized, and the mouse hybridoma cell strain 14C2 capable of efficiently secreting the anti-CK 8 protein monoclonal antibody and the anti-CK 8 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing CK8 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

Monoclonal antibody of anti-CK 8 protein, cell strain, preparation method and application thereof

Technical Field

The invention relates to the field of biomedical engineering, in particular to a monoclonal antibody for resisting CK8 protein, a cell strain, a preparation method and application thereof.

Background

Cytokeratin 8(Cytokeratin8, CK8) is localized to human chromosome 12q13 as one of important structures of cytoskeleton, and is expressed in simple epithelium, tubular epithelium and pseudostratified epithelium. CK8/18 is the major component of the intermediate fiber in the monolayer of epithelial cells, mainly in the form of dimers; it is distributed in the parts of gastrointestinal tract, liver, membrane gland, breast and other cancer prone parts. CK8 is specifically expressed in monolayer epithelial tissue, localized to the cytoplasm, and consists of 483 amino acids. Cytokeratin8 has 3 functional domains, including a centrally located, conserved, rod-like domain forming an a-helix, and attached thereto an NH 2-terminal head domain and a COOH-terminal tail domain. Where the rod-like domain consists of two helices, each of which can be further divided into two subregions, point mutations in the rod-like domain can lead to loss of cellular integrity and severe signs.

Cytokeratins are useful in maintaining the integrity of the cell structure and also in signal transduction and cell differentiation. Mutations in the transgene lead to cryptogenic cirrhosis. CK8 is expressed persistently in epithelial tumor cells of different origins and is a marker of its invasive properties. Research indicates that cell attachment blocks on cellulose are found by culturing mouse liver cells with CK8 knocked out and H4 liver cells of rats, and the slow movement indicates that CK8 plays an important role in development and tumorigenesis and is mainly realized by regulating adhesion and movement of epithelial cells. Researchers found that CK8/18 positivity was closely linked to the developmental diversity of liver tumors in B6C3F1 and C57B 1/6J-background mice. The structure of the dimer consisting of CK8 and CK18 is determined by CK8 squamous Ser73 and Ser431, and the tumorigenic transformation of mouse liver tissues can lead to positive alkalophilic staining, which shows that CK8 and CK18 are closely related to the induction of tumor cell proliferation. It has been shown that overexpression of cytokeratin CK8/18 may drive the process of hepatoma formation in rats with malignant transformation of precancerous cells GST-P positive foci. Recent studies have shown that CK8 is involved in the development of several liver diseases. CK8 knockout mice develop occasional bleeding and are more susceptible to liver damage. In chronic liver disease, CK8 is associated with the severity and progression of the disease.

Disclosure of Invention

The inventor provides an anti-CK 8 monoclonal antibody, which is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20769.

Further, the monoclonal antibody specifically recognizes the CK8 protein.

Further, the monoclonal antibody specifically recognizes the amino acid sequence shown as SEQ ID1 in the CK8 protein.

Further, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID 3.

Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown as SEQ ID 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown as SEQ ID 5.

Further, the monoclonal antibody is a mouse IgG3 subtype monoclonal antibody.

The inventor also provides a preparation method of the anti-CK 8 monoclonal antibody immunogen: selecting 340-365 amino acid sequence at the C terminal of CK8 protein as antigen peptide, and coupling the antigen peptide with KLH carrier protein to obtain the immunogen; the amino acid sequence of the antigen peptide is shown as SEQID 1.

The inventor also provides a hybridoma cell strain secreting anti-CK 8 protein molecules, wherein the cell strain is a mouse hybridoma cell strain 14C2, the cell strain is preserved in China general microbiological culture Collection center (CGMCC NO 20769) at 9 and 17 months of 2020, and the accession number is CGMCC NO 20769, and the cell strain is the institute of microbiology of China academy of sciences No. 3 of the North West Lu No. 1 Hopkins of the Korean-Yang district in Beijing.

The inventor also provides the application of the monoclonal antibody in CK8 protein immunoassay.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

Different from the prior art, the invention has the beneficial technical effects that: according to the technical scheme, the 340-365 amino acid sequence at the C terminal of CK8 protein is selected as an antigen peptide, and the antigen peptide is coupled with KLH carrier protein to obtain the immunogen; the amino acid sequence of the antigen peptide is shown as SEQ ID1, the mouse is immunized, and the mouse hybridoma cell strain 14C2 capable of efficiently secreting the anti-CK 8 protein monoclonal antibody and the anti-CK 8 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing CK8 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph of immunohistochemical staining results for lung adenocarcinoma; the left part is CK8 secreted by 14C2, and the right part is murine monoclonal antibody TS 1.

FIG. 2 is a graph showing the immunohistochemical staining results of breast ductal infiltration cancer; the left part is CK8 secreted by 14C2, and the right part is murine monoclonal antibody TS 1.

FIG. 3 is a graph comparing the results of IHC staining of the liver and bile duct epithelium; the left part is CK8 secreted by 14C2, and the right part is murine monoclonal antibody TS 1.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

Example 1 polypeptide Synthesis and chemical coupling to Carrier proteins

Sequence and secondary structure analysis is carried out according to the protein sequence with the accession number of P05787 in Uniprot, and the molecular weight of the CK8 protein with the full length of 483 amino acids is about 53 kDa. According to the parameters of secondary structure (secondary structure) and Surface Accessibility (Surface Accessibility) of the predicted protein by an online server http:// www.cbs.dtu.dk/services/NetSurfP/and the analysis result of the antigenicity index, the amino acid sequence at the 340-position 365 is selected as an antigenic peptide for chemical synthesis (the sequence is shown as SEQ ID1 in a sequence table), and a cysteine is added at the carboxyl terminal of the antigenic peptide for providing sulfhydryl coupling for facilitating coupling. The coupling steps are as follows:

the equine activated keyhole limpet hemocyanin kit from Thermo Scientific (cat # 77653) was selected and operated according to the protocol provided for the kit. For the antigenic peptide to be conjugated, the free thiol group of the antigenic peptide is first detected with Ellman's reagent (Thermo Scientific, cat # 22582): adding 100 mu L of Ellman reagent stock solution into a 96-well plate, adding 10 mu L of antigen peptide solution, measuring the ultraviolet absorption value of the solution by a Nano Drop spectrophotometer under the condition that the lambda is 412nm, and performing the next step if the OD value is more than 0.15; the OD value is less than 0.15 and more than 0.05, and antigen peptide is supplemented until the requirement is met; the OD value is less than 0.05, and the antigen peptide is returned to the synthesis step for quality control. To begin conjugation, 200. mu.L of deionized water was added to each mcKLH package to make up a 10mg/mL solution of KLH, 2mg of hapten was dissolved in 500. mu.L of Imject EDC coupling buffer, 500. mu.L of antigenic peptide solution was added to 200. mu.L of carrier protein solution, 1mL of deionized water was added to one package of EDC (10mg) and shaken slowly to dissolve completely, 50. mu.L of antigenic peptide solution was added to the mcKLH polypeptide solution and after 2 hours of reaction, unconjugated crosslinker and salts were removed by desalting column treatment.

The immunogen prepared by the method is crosslinked with Bovine Serum Albumin (BSA), and the obtained crosslinked product is named as BSA-CK8-PEP and is used for screening antibodies, determining affinity and analyzing immunoblotting.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The crosslinked polypeptide of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), 3 ICR mice (purchased from Beijing Wittinger laboratories, Inc.) were immunized, and each mouse was injected subcutaneously at 6-point in the abdomen at a dose of 20. mu.g/mouse. The booster was administered every 14 days and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20. mu.g/mouse. 7 days after 3 times of booster immunization, the titer of multiple antibodies of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by normal saline, and the dosage is 20 mu g/mouse.

Second, cell fusion

A mouse spleen cell suspension with qualified immunity is prepared aseptically, mixed with mouse myeloma cell sp2/0(ATCC number CRL-8287) at a ratio of 5:1, and centrifuged at 1500rpm for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1ml of PEG1500 (Roche) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, and centrifuged at 1000rpm for 5 min. After discarding the supernatant, 10ml of serum (PAA) was added to the supernatant, the cells were carefully blown up, 5ml of thymocytes mixed with 10XHAT (Sigma) was added, and the mixture was mixed well. Then, 25ml of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and poured into 20 cell culture dishes. The cell culture dish was placed in a wet box and incubated in a 5% CO2 incubator at 37 ℃.

Cloning and ELISA screening of positive hybridoma cells

The size and density of the clone cell mass are moderate 7 days after fusion, and the round, solid and large clone mass is sucked under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and the culture is carried out in a 5% CO2 incubator at 37 ℃. After 3 days, the cell mass was approximately 2/3 basal areas, and 100. mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely replaced and 200. mu.L of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates previously prepared in medium (containing feeder cells and HT). After five days, 100 μ l of supernatant was subjected to a third ELISA screening, and the positive clones were transferred to 6-well plates and cell culture flasks successively for expanded culture and frozen.

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

First, preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵And 1 ml. Intraperitoneal injection of suspended cells mice sensitized beforehand with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. The ascites fluid in the middle is carefully aspirated and collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

Secondly, purification of monoclonal antibody

Antibodies were purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography as described. Purity was assessed on SDS-PAGE gels and concentration was determined by Bradford method. Purified antibody was stored at-20 ℃.

EXAMPLE 4 characterization of monoclonal antibodies

First, subclass identification

Coated goat anti-mouse IgG (Beijing China fir Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5. mu.g/ml with 100. mu.l/well at 4 ℃ overnight in 100mM PBS (pH 7.4). The liquid was decanted, washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 h. The decanted solution was washed 3 times with PBS-T. Using a confining liquid 1: 1000 dilution of HRP-labeled goat anti-mouse (κ, λ) antibody or 1: HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibodies (Southern Biotech) were diluted at 2000 in 0.1ml per well and added to the appropriate wells, followed by incubation at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Add 50. mu.l of 0.15% ABTS (Southern Biotech) and 0.03% H per well₂O₂The reaction was performed in the citric acid buffer (pH4.0), and the OD value at a wavelength of 405nm was measured within 10 to 20 min.

The results show that the monoclonal antibody of the present invention is a murine monoclonal antibody of the IgG3 type.

Second, determination of affinity constant

The BSA-CK8-PEP conjugate prepared in example 1 was coated at a concentration of 2. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 2h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 3 was prepared from 1: 200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: 20000 dilution, 100 μ l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Adding 0.1% into 100 μ l of the solution per wellTMB (Sigma Co.) and 0.03% H₂O₂The reaction mixture was developed in a citric acid-phosphoric acid buffer for 10min, and 50. mu.l of a 0.5M sulfuric acid solution was added thereto to terminate the reaction. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. Drawing a curve of OD value corresponding to the dilution factor of the antibody, finding the dilution factor A corresponding to half of the maximum binding OD value, and calculating the affinity constant of the antibody to be 9.6 × 10 by using the following formula⁸。

Reaction specificity and application effect of monoclonal antibody

Taking the BSA-CK8-PEP coupled product prepared in example 1, detecting the recognition specificity of the monoclonal antibody of the invention by an immunoblotting method, wherein the immunoblotting experiment process is as follows: the BSA-conjugated polypeptides and the control BSA protein were loaded at about 5-10ng each and subjected to 12% polyacrylamide gel electrophoresis. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS-T blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibodies (1: 1000 dilution) of the antibody secreted by the 14C2 hybridoma were added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, add 1: a5000-diluted goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) was incubated at room temperature for 1 hour. Washing the membrane with TBST again, adding ECL (Beijing prilley Gene technology Co., Ltd.), and collecting chemiluminescence image data with ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

Example 5 determination of variable region sequences of antibodies

Collecting fresh hybridoma cells, collecting supernatant, performing antigen binding property verification to confirm that the cell strain for cloning can indeed secrete required antibody, and centrifuging to collect 10⁶The hybridoma cells described above. Total RNA from hybridoma cells was extracted by Trizol method, and 9. mu.L of total RNA was added with 2.5. mu.L of oligo (dT) 12-18 primer (10mM) and 5. mu.L of dNTPs, mixed well, incubated at 70 ℃ for 5 minutes and then placed on ice for 5 minutes, or denatured according to the reverse transcriptase used. Followed by the addition of 5 μ LRT buffer (5X), 2.5. mu.L of DTT (0.1M) and 1. mu.L of reverse transcriptase were reacted at 42 ℃ for 1 hour. The reaction was terminated by incubation at 70 ℃ for 15 minutes, and the obtained cDNA was stored at-20 ℃. The first strand cDNA thus obtained was subjected to PCR amplification, and 25pmol each of primers was added to a 50. mu.L reaction system, and the sequences of the primers for amplification of the heavy chain variable region and the light chain variable region were designed and synthesized based on the sequence of the murine monoclonal antibody primer as described in "recombinant antibody" by Hippo Seiyaku (science publishers, 2005 publications).

The rest dNTPs and buffer are added according to the conventional method, and finally 1 mu L of cDNA template and 1U of hot start Taq DNA polymerase are added. Setting PCR amplification program as 94 deg.c for 40 sec, 52 deg.c for 40 sec, 72 deg.c for 40 sec, 20-25 cycles, final extension at 72 deg.c for 3 min, and setting the product at 4 deg.c for use or direct electrophoresis. 20 μ L of PCR product was analyzed by electrophoresis, separated on 1.5% agarose gel, the length of light chain (. kappa.light chain) was between 320-340bp and the length of heavy chain was between 340-370bp, and the region-specific product was recovered by gel cutting and cloned into T-vector or expression vector for sequencing.

Example 6 immunohistochemical tissue chip staining and identification

Chip preparation process

HE sections were first stained for each sample to identify tumor sites. The tissue chip was produced using a fully automatic tissue chip machine of 3 DHISTECH. And putting the prepared tissue chip wax block into a wax block manufacturing mold, putting the mold into an oven at 68 ℃ for 10 minutes to enable the tissue core and the wax of the receptor wax block to be fused into a whole, then slightly taking the mold out of the oven, cooling the paraffin in a semi-molten state for about 30 minutes at room temperature, putting the mold into a refrigerator at-20 ℃ for freezing for 6 minutes, taking the tissue chip wax block out of the mold, and slicing or putting the tissue chip wax block into the refrigerator at 4 ℃ for storage for later use. And (3) trimming, continuously slicing, setting the thickness to be 3 mu m, floating the continuous slices in 40% alcohol, naturally unfolding, transferring the separated slices into warm water at 50 ℃ for 30 seconds, pasting the slices with a glass slide treated by polylysine, baking the prepared tissue chip in an oven at 68 ℃ for 2 hours, taking out, cooling at room temperature, and storing in a refrigerator at-4 ℃.

IHC staining and analysis

General ofXylene was dewaxed 3 times for 6 minutes each time, hydrated in 100%, 95%, 85% gradient ethanol for 3 minutes each time, and finally rinsed with tap water. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3 min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 15-30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds, PBS bluing for 30 seconds. Dehydration was carried out in a gradient of 85% (3 min) -95% (3 min) -100% (3 min) alcohol, finally xylene was transparent for 3 min, and the gel was blocked with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

Data statistics

1. Tumor tissue chip detection results:

the antibody CK8(14C2) and the commercial antibody CK8 (murine monoclonal antibody TS1) are synchronously detected on different human tumor tissue chips (comprising 25 cases of lung adenocarcinoma and 18 cases of breast ductal infiltration cancer) and the detection results are compared.

The immunohistochemical results of CK8 were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:

the result shows that the 14C2 cell strain secretes the anti-CK 8 protein monoclonal antibody with accurate staining positioning, clear staining, no non-specific staining and clean background. In the positive detection, 2 cases of lung adenocarcinoma and 2 cases of breast ductal infiltration cancer exist, the number of staining cells and staining intensity of CK8 secreted by 14C2 are higher than that of CK8 sold in the market, which indicates that the sensitivity is higher, and false negative results are effectively avoided.

FIG. 1 is a graph of the immunohistochemical staining results of lung adenocarcinoma (CK 8 secreted by 14C2 on the left and murine mAb TS1 on the right).

FIG. 2 is a graph of immunohistochemical staining results of breast ductal infiltration cancer (CK 8 secreted by 14C2 on the left, and murine monoclonal antibody TS1 on the right).

2. Detection results of the normal tissue chip:

the normal tissue chip comprises 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed operation specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

The antibody (14C2) and a commercial antibody (murine monoclonal antibody TS1) are synchronously detected on a normal tissue chip, and the negative and positive detection results are consistent, which shows that the specificity of the antibody in the normal tissue is equivalent to that of the commercial antibody.

FIG. 3 is a comparison graph of IHC staining results of liver bile duct epithelium (CK 8 secreted by 14C2 on the left, and murine monoclonal antibody TS1 on the right).

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

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Claims

1. An anti-CK 8 monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20769.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes CK8 protein.

3. The monoclonal antibody of claim 2, wherein the monoclonal antibody specifically recognizes a protein fragment represented by seq id1 in the CK8 protein.

4. The monoclonal antibody of claim 1, wherein the DNA sequence encoding the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID2, and the DNA sequence encoding the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID 3.

5. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown as SEQ ID 5.

6. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG 3-type monoclonal antibody.

7. A hybridoma cell strain capable of secreting anti-CK 8 protein molecules is a mouse hybridoma cell strain 14C2, is preserved in the China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 20769.

8. Use of the monoclonal antibody of any one of claims 1-6 in the preparation of a CK8 protein immunodetection reagent.

9. The use according to claim 8, wherein said immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.