CN113278070B - anti-CK 17 protein monoclonal antibody and cell strain, preparation method and application thereof - Google Patents

Abstract

The invention relates to a monoclonal antibody capable of recognizing human CK17 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. The technical proposal chemically synthesizes 410-432 amino acid at the C terminal of the CK17 protein as antigen peptide, and obtains immunogen after coupling KLH. The CK17-KLH protein is used for immunizing a mouse, and a mouse hybridoma cell strain 4D2 capable of efficiently secreting the anti-CK 17 protein monoclonal antibody and the anti-CK 17 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 CK17 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

anti-CK 17 protein monoclonal antibody, cell strain thereof, preparation method and application

Technical Field

The invention relates to the field of biomedical engineering, in particular to a monoclonal antibody for resisting CK17 protein, a cell strain, a preparation method and application thereof.

Background

Cytokeratins express different types of cytokeratins in the epithelial cells of different tissues or organs as well as at different stages of cell differentiation. The expression of cytokeratins in normal cervical tissues, and the change of some cytokeratins and the certain regularity in the process of transforming the normal cervical tissues into precancerous lesions or invasive tumors show that the cytokeratins have obvious molecular diversity, such as: colorectal adenocarcinoma is usually CK20 positive and CK7 negative; in the process of generating basal skin tumor, CK17 can play a role in self immune regulation, thereby promoting the proliferation of epithelial cells and the growth of tumor.

During the transformation of normal cervical tissues to cervical cancer and precancerous lesions, the expression of CKs is changed in series and shows certain regularity, especially the expression of CK 17. The results of Smedts et al showed that CK17 expression increased gradually as the lesion changed from CIN to invasive carcinoma, and thus it was suggested that the lesion expressed CK17 had the potential to progress, and that no expression had the potential to resolve or persist but not progress. Carrilho et al studied 64 patients, 10 of them, and 44 of them had cervical infiltrates, and as a result, the positive expression rates of CK17 were 0%, 40%, and 73.2%, respectively, indicating that as the lesion progressed, the expression of CK17 increased, and CK17 was considered as a specific marker of cervical malignancy. Ikeda et al studied 134 patients, including CIN I39, CIN II 31, CIN III 43 and invasive carcinoma 21, and found positive expression rates of CK17 of 33.3%, 58.1%, 81.4% and 95.2%, respectively, and CK17 positivity was associated with increased CIN grade, suggesting that CK17 may play a role in screening high-risk cases of cervical cancer. The results of the studies by the Wang Shiminmen et al show that the positive expression rate of CK17 is gradually increased along with the gradual increase of lesion degree from the normal cervix → CIN I → CIN II → CIN III → cervix carcinoma, and the positive expression rate of CK17 is higher as the lesion degree of the cervix carcinoma is higher. Therefore, it is considered that the future development trend of cervical lesions, namely cervical lesions expressing CK17, is predicted by the expression of CK17, and on the contrary, cervical lesions not expressing CK17 are less likely to develop into cervical cancer in the future.

In addition, studies have shown that CK17 is associated with the clinical pathological features of Oral Squamous Cell Carcinoma (OSCC) patients, and that high expression of CK17 is significantly associated with prognosis of oral squamous cell carcinoma patients, particularly whether lymph node metastasis is significantly associated, the risk of lymph node metastasis increases 6-fold when CK17 is positively expressed, and in patients undergoing surgery and radiotherapy, low expression of CK17 is an independent marker of early disease recurrence and disease-specific death, and the risk of disease recurrence increases about 4-fold in patients with low expression of CK17 over the risk of high expression of K17. The research result shows that: CK17 and CK19 are likely to be potential tumor markers for predicting the prognosis of OSCC patients.

Disclosure of Invention

The inventor provides an anti-CK 17 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown as SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

Furthermore, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 3.

Further, the monoclonal antibody specifically recognizes the CK17 protein.

Further, the monoclonal antibody is mouse IgG_2aSubtype monoclonal antibodies.

Furthermore, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20762.

The inventor also provides a preparation method of the anti-CK 17 protein monoclonal antibody, which comprises the steps of selecting an amino acid sequence (from the 410 th site to the 432 th site) shown by SEQ ID NO.1 in CK17 protein, adding a cysteine at the carboxyl terminal of the amino acid sequence, and coupling the amino acid sequence with a carrier protein KLH to serve as immunogen.

The inventor also provides a hybridoma cell strain secreting anti-CK 17 protein molecules, wherein the cell strain is a mouse hybridoma cell strain 4D2 which is preserved in China general microbiological culture Collection center (CGMCC NO 20762) at 9 and 17 months of 2020, and the accession number is CGMCC NO 20762, and the cell strain is the institute of microbiology of China academy of sciences No.3 of North West Lu No.1 Hopkins of the Korean-Yang district in Beijing.

The inventor also provides the application of the monoclonal antibody in CK17 protein immunoassay.

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

The inventor finally provides a CK17 protein immunohistochemical detection reagent, wherein the immunohistochemical detection reagent contains an amino acid sequence of which the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO. 4; the monoclonal antibody of anti-CK 17 protein with the amino acid sequence of the light chain variable region as shown in SEQ ID No.5 as the effective component.

Different from the prior art, the invention has the beneficial technical effects that: according to the technical scheme, the 410 th to 432 th amino acids at the C terminal of CK17 protein are selected as antigen peptides, the immunogen obtained after coupling KLH is used for immunizing a mouse, and a mouse hybridoma cell strain 4D2 capable of efficiently secreting the anti-CK 17 protein monoclonal antibody and the anti-CK 17 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 CK17 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph comparing immunohistochemical staining results for esophageal squamous cell carcinoma; the left is CK17 secreted by 4D2, and the right is commercial CK17 (E3).

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 preparation of immunogen

First, immunogen preparation

Sequence and secondary structure analysis is carried out according to the protein sequence with the accession number of Q04695 in Uniprot, and the molecular weight of the CK17 protein with the total length of 432 amino acids is about 48 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/through the results of analysis of its antigenic index (Jameson BA, Wolf H. the antigenic index: a novel algorithm for predicting the antigenic definitions. composite biosci.1988,4(1):181-6.), the amino acid sequence SEQ ID NO.1 VEEVQKSRVISIEQVHQTTR at position 410 and 432 is selected as the antigenic peptide for chemical synthesis, and for coupling, a cysteine is added at the carboxyl end of the polypeptide for providing thiol coupling.

Coupling and purification of polypeptide

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 polypeptide to be conjugated, the free thiol group in the polypeptide 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 polypeptide 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 the polypeptide is added until the requirement is met; and the OD value is less than 0.05, and the polypeptide is returned to the polypeptide 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 polypeptide 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 polypeptide solution was added to mcKLH polypeptide solution and after 2 hours of reaction, unconjugated crosslinker and salts were removed by desalting column treatment to give CK 17-KLH.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The cross-linked polypeptide of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 2 SPF-grade BALB/c female mice and 2 ICR mice (purchased from Beijing Wintolite laboratory technologies, Inc.) were immunized and injected ventrally subcutaneously at 6 spots per mouse at a dose of 60. mu.g/mouse. The booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co., F5506) at a dose of 30. 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 50 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. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

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

Preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵And 1 ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. Carefully aspirate the middle ascites collectionStoring in a centrifuge tube at 4 deg.C or-20 deg.C.

Secondly, purification of monoclonal antibody

The antibody was purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography according to the instructions. 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 2000 by 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 invention is IgG_2aType murine monoclonal antibodies.

Second, determination of affinity constant

CK17 polypeptide 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 purified 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. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The mixture was developed in a citric acid-phosphoric acid buffer for 10min, and 50. mu.l of 0.5M sulfur was addedThe acid solution stops 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

The CK17 polypeptide prepared in example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, and 12% polyacrylamide gel electrophoresis was performed. 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 to the antibody secreted by the 4D2 hybridoma (1: 1000 dilution) 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 of 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. Then, 5. mu.L of RT buffer (5X), 2.5. mu.L of DTT (0.1M) and 1. mu.L of reverse transcriptase were added and 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 obtained was subjected to PCR amplification, and each primer was added to a 50. mu.L reaction system25pmol of primer sequences for amplification of heavy chain variable region and light chain variable region were designed and synthesized based on the sequence of the murine mAb primer in the book "recombinant antibodies" (published by scientific Press, 2005) compiled by Higgera.

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

Conventional xylene dewaxing was performed 3 times for 6 minutes each, 100%, 95%, 85% gradient ethanol hydration for 3 minutes each, and finally tap water rinse. 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 CK17(4D2) and the commercially available antibody CK17(E3) were simultaneously detected on tumor tissue chips (23 cases of cervical cancer and 15 cases of squamous cell lung cancer), and the detection results were compared.

The immunohistochemical results of CK17 were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:

the result shows that the monoclonal antibody of the anti-CK 17 protein secreted by the 4D2 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In the positive detection, 2 cases of cervical cancer and 3 cases of esophageal squamous carcinoma exist, and the number of staining cells and the staining intensity of CK17 antibody secreted by 4D2 are obviously higher than that of CK17 sold in the market; the CK17 antibody secreted by the 4D2 is higher in sensitivity, and false negative results are effectively avoided.

FIG. 1 is a graph comparing immunohistochemical staining results for esophageal squamous carcinoma (CK 17 secreted from 4D2 on the left and CK17 on the market 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 (4D2) and the commercial antibody (E3) were detected on the normal tissue chip synchronously, and the negative and positive detection results were consistent, indicating that the specificity of the antibody in the normal tissue was equivalent to that of the commercial antibody.

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 scope of the present invention is not limited thereby. 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 (9)

1. The monoclonal antibody for resisting the CK17 protein is characterized in that the amino acid sequence of a heavy chain variable region of the monoclonal antibody is the amino acid sequence shown as SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

2. The monoclonal antibody according to claim 1, wherein the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No.2, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No. 3.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes CK17 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG_2bSubtype monoclonal antibodies.

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 20762.

6. A hybridoma cell strain capable of secreting anti-CK 17 protein molecules is a mouse hybridoma cell strain 4D2, is preserved in the China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 20762.

7. Use of the monoclonal antibody of any one of claims 1-5 in the preparation of a CK17 protein immunodetection reagent.

8. Use according to claim 7, characterized in that said immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assays.

9. An immunohistochemical detection reagent for CK17 protein, comprising the anti-CK 17 monoclonal antibody of claim 1 as an active ingredient.

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