CN113061184A

CN113061184A - Monoclonal antibody of anti-CK 7 protein, cell strain, preparation method and application thereof

Info

Publication number: CN113061184A
Application number: CN202110447827.7A
Authority: CN
Inventors: 陈昌星; 杨清海; 陈惠玲; 王小亚; 周洪辉
Original assignee: Fuzhou Maixin Biotechnology Development Co ltd
Current assignee: Fuzhou Maixin Biotechnology Development Co ltd
Priority date: 2021-04-25
Filing date: 2021-04-25
Publication date: 2021-07-02
Anticipated expiration: 2041-04-25
Also published as: CN113061184B

Abstract

The invention relates to a monoclonal antibody capable of identifying human CK7 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. The technical proposal selects 8 groups of amino acid peptide segments (1-16, 74-86, 139-150, 324-335, 347-357, 401-410, 421-434 and 444-469 sites) at the C terminal and the N terminal of the CK7 protein to be connected in series for codon optimization to form the CK7 recombinant peptide segment suitable for expression in Escherichia coli BL21(DE3), and the finally obtained recombinant protein comprises a GST protein tag, a CK7 recombinant peptide segment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 25C4 capable of efficiently secreting the anti-CK 7 protein monoclonal antibody and the anti-CK 7 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning.

Description

Monoclonal antibody of anti-CK 7 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 CK7 protein, a cell strain, a preparation method and application thereof.

Background

CK is a water-soluble, polypeptide chain encoded by the cytokeratin gene. It is the most complex intermediate filament protein that is part of the cytoskeletal protein. Maintaining the continuity and integrity of epithelial tissue is a major biological function of cytokeratins. Up to 20 or more cytokeratins have been identified so far. Cytokeratins can be further classified into type I and type II according to their different pH values. The type I cytokeratin is slightly acidic and has small relative molecular mass, and mainly comprises CK 9-CK 20; type II cytokeratins are basic and have large relative molecular mass, and mainly comprise CK 1-CK 8. The cytokeratin isoforms expressed in different epithelial tissues are also different. The expression of cytokeratins in epithelial cells is regulated by the degree of cell differentiation, and the expressed cytokeratin subtypes of epithelial cells of different origins are different. When normal epithelial cells are malignant, the expressed cytokeratins are not changed. This feature helps identify the source of the tumor tissue and accurately classify epithelial tumors. Currently, more and more tumor markers are used for the detection of tumor micrometastases.

CK7 is a basic protein in the CK family, has a relative molecular mass of about 54000, belongs to a low molecular mass protein, and codes for a gene located on chromosome 12 of human. It is mainly expressed in glandular epithelium and transitional epithelium, and can be localized in cytoplasm, mitochondria and nucleus. CK7 forms a complex network system in cytoplasm by combining with other accessory proteins, and maintains the morphological integrity and continuity of epithelial cells. The CK7 still retains its expression pattern in normal tissues in epithelial tumors and metastases thereof, thus having important roles in immunohistochemical differential diagnosis of tumors, metastasis, infiltration and classification of tumors, subtype determination and the like. Most of the adenocarcinoma tissues CK7 were expressed positively, and the expression rate thereof was reported to be 96% in lung adenocarcinoma, 95% in pancreatic cancer, 86% in ductal breast carcinoma and 95% in lobular breast carcinoma. CK7 is a marker for epithelial-derived tumor cells and is not expressed in mesenchymal tissues such as lymph nodes and blood. When CK7 is expressed in tissues such as lymph nodes, it is considered that micrometastases of tumors exist.

Disclosure of Invention

The inventor provides an anti-CK 7 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 CK7 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 20776.

The inventor also provides a preparation method of the anti-CK 7 protein monoclonal antibody, an antigen used for immunizing a mouse is recombinant protein, the recombinant protein is expressed by escherichia coli in a recombinant mode, and the recombinant protein comprises a GST protein tag, a CK7 recombinant peptide segment and a histidine protein tag; the amino acid sequence of the CK7 recombinant peptide segment is the amino acid sequence shown in SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting anti-CK 7 protein molecules, wherein the cell strain is a mouse hybridoma cell strain 25C4, the cell strain is preserved in China general microbiological culture Collection center (CGMCC NO 20776) at 9 and 17 months of 2020, and the preservation number is CGMCC NO 20776, and the address 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 CK7 protein immunoassay.

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

The inventor finally provides a CK7 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 7 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: the technical proposal selects 8 groups of amino acid peptide segments (1-16, 74-86, 139-150, 324-335, 347-357, 401-410, 421-434 and 444-469 sites) at the C terminal and the N terminal of the CK7 protein to be connected in series for codon optimization to form the CK7 recombinant peptide segment suitable for expression in Escherichia coli BL21(DE3), and the finally obtained recombinant protein comprises a GST protein tag, a CK7 recombinant peptide segment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 25C4 capable of efficiently secreting the anti-CK 7 protein monoclonal antibody and the anti-CK 7 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 CK7 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph of purified pectin from CK7 recombinant peptide fragment fused with histidine tag in example 1, wherein M represents Marker; 1 is a supernatant sample after ultrasonic treatment; and 2 is a post-sonication sedimentation sample.

FIG. 2 is a graph comparing the results of immunohistochemical staining for breast cancer; the left part is CK7 secreted by 25C4, and the right part is commercial CK 7.

FIG. 3 is a graph comparing immunohistochemical staining results of placental trophoblast cells; the left part is CK7 secreted by 25C4, and the right part is commercial CK 7.

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 recombinant CK7 protein fragment

First, gene optimization and synthesis

CK7 selects CK7 recombinant peptide segments in which 8 specific peptide segments (1-16, 74-86, 139-150, 324-335, 347-357, 401-410, 421-434, 444-469AA) are connected in series according to the protein sequence with the accession number P08729 in the Uniprot database; the amino acid sequence of the CK7 recombinant peptide segment is the amino acid sequence shown in SEQ ID NO. 1; directly optimized into a gene fragment suitable for being expressed in Escherichia coli BL21(DE 3). EcoR I and XhoI cleavage sites were added at the 5 'and 3' ends of the gene, respectively, during PCR.

And separating the PCR product by agarose gel electrophoresis, recovering, carrying out EcoR I and XhoI enzyme digestion on the recovered fusion protein gene and a plasmid vector Pet30a-GST for expression, carrying out electrophoresis recovery again, and connecting by T4 DNA ligase. The ligation product was transformed into E.coli competent cells BL21(DE3), and the colonies on the plate were picked and inoculated for PCR identification of the bacterial suspension. And selecting clones with positive PCR results for sequencing analysis, and using the clones with completely correct sequences.

The selection of different antigens for immunization makes it possible to prepare antibodies with different binding characteristics, the molecules present simultaneously a plurality of variants due to variable cleavage, and finally the recognition ability and pattern of different antibodies on antigen-expressing cells are different. The CK7 molecule is analyzed according to published sequence, according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure on cell membrane, a region which is suitable for soluble expression and has good immunogenicity is selected for recombinant expression, 8 specific peptide segments (1-16, 74-86, 139-150, 324-335, 347-357, 401-410, 421-434, 444-469AA respectively) of CK7 are selected to be added in series (the peptide segments are separated by adding amino acids), codon optimization is carried out, and the molecular weight of the obtained CK7 recombinant peptide segment is about 42 kDa. The CK7 recombinant peptide segment is obtained by using prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of CK7 recombinant peptide with antigenicity and protein tags for recombinant protein purification, wherein the protein tags are GST and HIS.

II, protein expression and purification

The overnight strain cultured by a single colony is transferred to 100mL LB culture medium according to the proportion of 1:100, kanamycin with the final concentration of 10 mug/mL is added, shaking culture is carried out at 37 ℃ until OD600 is 0.6-0.8, 1mmol/L IPTG is added, shaking culture is carried out at 16 ℃ for overnight, and the strain is collected and then is crushed by ultrasound. The recombinant protein is provided with a histidine tag, and affinity purification of the protein is performed by using a nickel column. Elution was carried out with 500mmol/L imidazole and SDS PAGE separation was carried out.

FIG. 1 is a graph of purified pectin of recombinant CK7 protein fused with histidine tag. The protein concentration is 0.5mg/mL, and the protein can be used for requirements of animal immunization and antibody screening and identification.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The CK7 recombinant peptide fragment prepared in example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), 4-6 week-old female ICR mice (purchased from Beijing Wintolite laboratory animals technologies, Ltd.) were immunized, and 6 spots of 20. mu.g/mouse were injected subcutaneously into the abdomen. 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 50 mu g/mouse.

Second, cell fusion

Sterile preparation of mouse spleen cell suspension with up-to-standard immunityIt was 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

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. 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. 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 resulting mixture was subjected to a color reaction in a 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

The CK7 recombinant peptide fragment 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. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The 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. The OD value is plotted against the dilution factor of the antibody, and the dilution factor A corresponding to half of the maximum binding OD value is found and calculated by the following formulaThe affinity constant of the antibody was calculated to be 3.84X 10⁹。

Reaction specificity and application effect of monoclonal antibody

The CK7 recombinant peptide fragment prepared in example 1 was taken, the recognition specificity of the monoclonal antibody of the present invention was detected 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 25C4 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 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

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, and adding primary antibody (the dilution ratio of the antibody is designed according to the antibody concentration in the first dilution) diluted in a proper proportion dropwise to incubate at room temperature (25 ℃)And (3) incubating for 1 hour, washing with PBS for 3 × 3 minutes, adding secondary antibody dropwise, incubating for 15-30 minutes at room temperature, washing with PBS for 3 × 3 minutes, throwing off PBS, and developing with a freshly prepared DAB developing solution for 3-10 minutes. 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 CK7(25C4) and the commercial antibody CK7(OV-TL 12/30) were tested simultaneously on different human tumor tissue chips (including 40 cases of breast cancer, 35 cases of ovarian mucinous adenocarcinoma and 100 cases of various tumor collective chips) and the test results were compared.

The immunohistochemical results of CK7 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 7 protein secreted by the 25C4 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In the immunohistochemical detection, the positive rate was comparable to the commercial antibody, but the positive intensity was higher than the commercial antibody. In 2 cases of breast cancer and 1 case of mucinous adenocarcinoma, the staining intensity of CK7 secreted by the 25C4 cell line is obviously higher than that of the CK7 sold on the market, which indicates that the sensitivity is higher and the false negative result is effectively avoided.

FIG. 2 is a comparison of immunohistochemical staining results for breast cancer (CK 7 secreted from 25C4 on the left and CK7 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 CK7(25C4) and the commercial CK7 antibody (commercial OV-TL 12/30) 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 of the immune combined staining results of placental trophoblast cells (CK 7 secreted at the left by 25C4 and CK7 commercially available at 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. The monoclonal antibody for resisting the CK7 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 CK7 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG_2aSubtype monoclonal antibodies.

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 20776.

6. The preparation method of the anti-CK 7 protein monoclonal antibody is characterized in that an antigen used for immunizing a mouse is recombinant protein, the recombinant protein is expressed by escherichia coli in a recombinant mode, and the recombinant protein comprises a GST protein tag, a CK7 recombinant peptide segment and a histidine protein tag; the amino acid sequence of the CK7 recombinant peptide segment is the amino acid sequence shown in SEQ ID NO. 1.

7. A hybridoma cell strain capable of secreting anti-CK 7 protein molecules is a mouse hybridoma cell strain 25C4, is preserved in the China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 20776.

8. Use of the monoclonal antibody of any one of claims 1-5 in an immunoassay for the CK7 protein.

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

10. An immunohistochemical detection reagent for CK7 protein, comprising the anti-CK 7 monoclonal antibody of claim 1 as an active ingredient.