Anti-CK56 antibody and application thereof

Abstract

The invention discloses an anti-CK5amp antibody and a preparation method of the anti-CK5amp antibody. The invention discloses an antibody 6 and application thereof, the antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of CDR-H1 of the heavy chain variable region is as shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 of the heavy chain variable region is as shown in SEQ ID NO: 5, and the amino acid sequence of CDR-H3 of the heavy chain variable region is as shown in SEQ ID NO: 6; the amino acid sequence of the CDR-L1 of the light chain variable region is as shown in SEQ ID NO: 7, the amino acid sequence of the CDR-L2 of the light chain variable region is as shown in SEQ ID NO: 8, and the amino acid sequence of the CDR-L3 of the light chain variable region is as shown in SEQ ID NO: 9. The anti-CK5amp antibody is a CK5amp antibody; the 6 antibody is a rabbit monoclonal antibody, the obtained antibody is high in titer, clear in specific positioning in immunohistochemical staining, free of non-specific background staining, wide in application and capable of being used in Western blot and flow cytometry experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody development, and specifically relates to an anti-CK5&6 antibody and an application thereof. Background Art

[0002] CK5&6 refers to cytokeratin 5 and cytokeratin 6, which are members of the cytokeratin family and play a major role in the cytoskeleton of epithelial cells, providing structural support and resisting mechanical stress for cells. In normal tissues, CK5&6 is mainly expressed in the basal cells of squamous epithelium and ductal epithelium, as well as some squamous epithelial germinal layer cells, myoepithelial cells, and mesothelial cells, but is usually negative in glandular epithelial cells.

[0003] Cytokeratin CK5&6 plays an important role in assisting diagnosis, judging the progression of the disease and prognosis in cancer: For patients with cancers such as breast cancer and lung cancer, since tumor cells produce a large amount of cytokeratin and release it into the surrounding tissues, the expression of cytokeratin 5 / 6 may increase. The detection of cytokeratin CK5&6 can help determine whether there are cancerous lesions and help further evaluate the patient's disease status. In addition, cytokeratin CK5&6 can also be used to predict the survival time and prognosis of cancer patients. Studies have shown that patients with high expression of cytokeratin CK5&6 are generally more likely to metastasize and relapse than those with low expression, and have a poorer prognosis.

[0004] Detection of cytokeratin CK5&6 by immunohistochemistry has important clinical significance in cancer diagnosis and treatment. It can not only be used for auxiliary diagnosis of various diseases, but also guide subsequent treatment decisions. Therefore, the development of anti-CK5&6 antibodies will help in auxiliary diagnosis of related diseases including tumors. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-CK5&6 antibody and its application. To this end, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides an anti-CK5&6 antibody, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region CDR-H1 is shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 6; the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 9.

[0006] In some embodiments of the present invention, the antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the following polypeptide sequences using Kabat online database analysis.

[0007] (1) A CDR-H1 comprising at least 5 amino acid sequences; the sequence is NYAMG (SEQ ID NO: 4).

[0008] (2) A CDR-H2 comprising at least 16 amino acid sequences; the sequence is IIRNDRNTYYASWAKG (SEQ ID NO: 5).

[0009] (3) A CDR-H3 comprising at least 11 amino acid sequences; the sequence is GGCADCITANI (SEQ ID NO: 6).

[0010] (4) A CDR-L1 comprising at least 11 amino acid sequences; the sequence is QASQSISSYLN (SEQ ID NO: 7).

[0011] (5) A CDR-L2 comprising at least 7 amino acid sequences; the sequence is RASTLAS (SEQ ID NO: 8).

[0012] (6) A CDR-L3 comprising at least 12 amino acid sequences; the sequence is QQTYNYNNVDNL (SEQ ID NO: 9).

[0013] In some embodiments of the present invention, the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:2, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:3.

[0014] In some embodiments of the present invention, the antibody is a rabbit anti-human monoclonal antibody, and the rabbit immunogen comprises the amino acid sequence shown in SEQ ID NO:1.

[0015] In some embodiments of the present invention, a polypeptide fragment synthesized from aa231-aa245 DSIVDERGRLDSELR (SEQ ID NO: 1) in the amino acid sequence of human CK5 protein (Uniprot ID: P13647) is selected as the immunogen sequence for preparing the antibody. This sequence is consistent with the amino acid aa226-aa240 sequence in CK6A protein, Uniprot ID: P02538. This polypeptide sequence is of low molecular weight. To ensure the immune effect, one amino acid C, i.e., cysteine, is added to the left side of the polypeptide sequence for cross-linking of macromolecule KLH. The aforementioned polypeptide fragment is cross-linked with macromolecule KLH to form a complete antigen.

[0016] A nucleic acid encoding the antibody described in the first aspect. By isolating the nucleic acid encoding the antibody described in the first aspect, the antibody can be produced in a recombinant manner, the nucleic acid is isolated and inserted into a replicable vector, and then further cloned or further expressed. Based on this, the second aspect of the present invention provides an expression vector comprising the nucleic acid described above. The sequence of the nucleic acid encoding the heavy chain variable region and the light chain variable region of the present invention can be changed, and such changes include the addition, deletion or non-conservative / conservative substitution of nucleotides. The DNA encoding the antibody can be easily isolated or synthesized using conventional procedures. A variety of vectors are available, and the vector components generally include but are not limited to one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0017] Preferably, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0018] The third aspect of the present invention provides a host cell, comprising the expression vector described above. In some specific embodiments of the present invention, the expression vector is a prokaryotic expression vector, and the host cell is a prokaryotic cell; in other specific embodiments, the expression vector is a eukaryotic expression vector, and the host cell is a eukaryotic cell. In a specific embodiment of the present invention, the expression vector is an Escherichia coli expression vector, and the host cell is a HEK293 cell.

[0019] The fourth aspect of the present invention provides the use of the antibody described in the first aspect in the preparation of an immunoassay tool for detecting and identifying CK5&6 proteins.

[0020] In some embodiments of the present invention, the immunoassay tool is a reagent, a kit, a chip or a test paper.

[0021] In some embodiments of the present invention, the immunodetection tool is used for at least one of immunohistochemistry, flow cytometry, and immunoblotting.

[0022] The fifth aspect of the present invention provides the use of the antibody described in the first aspect in the preparation of a lung cancer type diagnostic kit for lung squamous cell carcinoma and lung adenocarcinoma. Through immunohistochemical staining, anti-CK5&6 rabbit monoclonal antibodies have brown cytoplasmic positive localization in tumor cells of lung squamous cell carcinoma tumor tissue, and are negative in tumor cells of lung adenocarcinoma tumor tissue.

[0023] The sixth aspect of the present invention provides a kit for detecting and identifying CK5&6 proteins, comprising the antibody described in the first aspect.

[0024] The seventh aspect of the present invention provides a method for obtaining the antibody of the first aspect, comprising: (1) culturing the host cell described in the third aspect, and (2) Isolating and purifying the antibody from the cultured cells.

[0025] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: the anti-CK5&6 antibody provided by the present invention has high titer, clear specific localization after immunohistochemical staining verification, and no non-specific background staining. The antibody can be used not only in pathological immunohistochemical staining experiments, but also in Western blot, immunoprecipitation and flow cytometry experiments, and has a wide range of uses.

[0026] Anti-CK5&6 antibodies can also be used in the diagnosis of lung cancer types: to distinguish between squamous cell carcinoma of the lung and adenocarcinoma of the lung; by immunohistochemical staining, anti-CK5&6 rabbit monoclonal antibodies have brown cytoplasm-positive localization in tumor cells of squamous cell carcinoma of the lung and are negative in tumor cells of adenocarcinoma of the lung. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The ELISA binding curve of the antigen in Example 1 of the present invention is shown; Figure 2 The results of immunohistochemical staining of different tissue sections with anti-CK5&6 rabbit monoclonal antibodies are shown; Figure 3 The results of Western blot detection of anti-CK5&6 rabbit monoclonal antibodies are shown; in the figure, 1 is a 1:500 dilution, 2 is a 1:1000 dilution, 3 is a 1:2000 dilution, and 4 is a 1:5000 dilution; Figure 4 The results of flow cytometry detection with anti-CK5&6 rabbit monoclonal antibodies are shown; the peak on the left indicates that the isotype control antibody does not recognize the CK5&6 protein on the A431 wild cell line, and the peak on the right indicates that the anti-CK5&6 rabbit monoclonal antibody can recognize the CK5&6 protein on the A431 wild cell line. DETAILED DESCRIPTION

[0028] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0030] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The instruments and equipment used in the following embodiments are all conventional laboratory instruments and equipment unless otherwise specified; the test materials used in the following embodiments are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0031] Example 1 Anti-CK5&6 monoclonal antibodies and their preparation A polypeptide fragment synthesized from aa231-aa245DSIVDERGRLDSELR (SEQ ID NO: 1) in the amino acid sequence of human CK5 protein (Uniprot ID: P13647) was selected as the immunogen sequence for the preparation of the antibody. This sequence is consistent with the amino acid aa226-aa240 sequence in the CK6A protein, Uniprot ID: P02538. This polypeptide sequence is of low molecular weight. To ensure the immune effect, an amino acid C, i.e., cysteine, was added to the left side of the polypeptide sequence for cross-linking of macromolecule KLH. The aforementioned polypeptide fragment cross-links macromolecule KLH to form a complete antigen.

[0032] The immunogen was used to immunize New Zealand white rabbits, and the steps were as follows: First, the polypeptide immunogen was coupled to the KLH macromolecular protein, and New Zealand white rabbits were immunized after dialysis. The first immunization was performed with 1 mg / mL, and 0.5 mL was mixed with an equal amount of complete Freund's adjuvant for the first immunization; 2 weeks later, 0.5 mg / mL, 0.5 mL was mixed with an equal amount of incomplete Freund's adjuvant for the second immunization, and the third and fourth immunizations were performed every 2 weeks according to the second immunization method.

[0033] 4. Blood was collected after immunization to test serum titer. After the titer was qualified, B cells of qualified rabbits were taken for freezing; B cells were obtained by the method described in our company's patent: CN201610724209.1. Then the cells were lysed by the method described in our company's patent: CN201910004299.0, and mRNA was extracted for RT-PCR. After RT-PCR, the obtained cDNA was subjected to PCR to obtain the light chain DNA and heavy chain DNA of the antibody. The light chain DNA and heavy chain DNA were connected to the pBV vector and transformed into competent cells, and cultured on a plate coated with culture medium for 12 hours. The cultured colonies were picked and shaken for culture, and the heavy chain plasmid and light chain plasmid were extracted after 24 hours of culture. The extracted light and heavy chain plasmids were transfected into HEK293 cells and cultured for 6 days.

[0034] After 6 days, the cell culture supernatant was harvested and the rabbit monoclonal antibody was obtained after purification.

[0035] The purified rabbit monoclonal antibody was sequenced.

[0036] The amino acid sequence of the H chain (heavy chain variable region) of CK5&6 rabbit monoclonal antibody (SEQ ID NO: 2) is as follows: ETGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLNNYAMGWVRQAPGKGLEYIGIIRNDRNTYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARG GCADCITANIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKT VAPSTCSKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPI EKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK The amino acid sequence of the L chain (heavy chain variable region) of CK5&6 rabbit monoclonal antibody (SEQ ID NO: 3) is as follows: DTRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTQFTLTISGVECADAAAYYCQQTYNYNN VDNLFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC The obtained anti-CK5&6 antibodies were analyzed using the Kabat online database; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are as follows: (1) a CDR-H1 of at least 5 amino acids; the sequence is NYAMG (SEQ ID NO: 4); (2) a CDR-H2 of at least 16 amino acids; the sequence is IIRNDRNTYYASWAKG (SEQ ID NO: 5); (3) a CDR-H3 of at least 11 amino acids; the sequence is GGCADCITANI (SEQ ID NO: 6); The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are as follows: (1) a CDR-L1 of at least 11 amino acids; the sequence is QASQSISSYLN (SEQ ID NO: 7); (2) a CDR-L2 of at least 7 amino acids; the sequence is RASTLAS (SEQ ID NO: 8); (3) A CDR-L3 of at least 12 amino acids; the sequence is QQTYNYNNVDNL (SEQ ID NO: 9).

[0037] Example 2 Enzyme-linked immunosorbent assay of anti-CK5&6 monoclonal antibodies The antibody can specifically bind to the immunogenic synthetic polypeptide sequence designed for human CK5&6.

[0038] The procedure was carried out according to the conventional iELISA method. The steps were as follows: (1) Coating: Use a synthetic peptide designed to specifically bind to human MUM1 for coating, 1 µg / mL, 50 µL / well, overnight at 4°C.

[0039] (2) Blocking: The next day, wash the plate three times with PBST and then add 5% skim milk for blocking at 100 µL / well at 30°C for 1 h.

[0040] (3) Primary antibody incubation: After washing the plate, the anti-MUM1 rabbit monoclonal antibody was diluted 4-fold from 1 µg / mL, with a total of 7 gradients. The diluted antibody was added to the ELISA plate at 50 µL / well and incubated at 30°C for 1 h.

[0041] (4) Secondary antibody incubation: After washing the plate, add 1:30,000 diluted HRP-labeled goat anti-rabbit IgG (Jackson, Cat#111-035-144) to the ELISA plate at 50 µL / well and incubate at 30°C for 40 min-60 min.

[0042] Color development and termination: After washing, add TMB color development solution at 50µL / well and react at 30℃ in the dark for 15min. After the reaction, add 50µL 1M H2SO4 to each well to terminate the reaction and read the OD450 absorbance value on the microplate reader. Prepare the standard curve based on the reading value. See the ELISA binding curve for details. Figure 1 .

[0043] The results showed that the readings at different dilutions were significantly different. The above antibodies could specifically bind to the immunogenic synthetic peptide sequences designed for human CK5&6. The ELISA binding curves of the CK5&6 monoclonal antibodies were as follows: Figure 1 As shown, the EC50 was 5.04 ng / mL.

[0044] Example 3 Application of anti-CK5&6 monoclonal antibodies in pathological immunohistochemistry Tissue sections of normal tonsil tissue, normal esophageal tissue, lung squamous cell carcinoma tumor tissue, and lung adenocarcinoma tumor tissue were selected for pathological immunohistochemical staining, and the tissue sections were subjected to the immunohistochemical staining experimental steps according to the conventional manual immunohistochemical operation process; after the immunohistochemical staining was completed, the sections were placed under a microscope for reading and photographing.

[0045] The immunohistochemical staining pictures taken under the microscope are shown in Figure 2 .

[0046] The results can be clearly seen from the pictures: anti-CK5&6 rabbit monoclonal antibodies have brown cytoplasm-positive localization in the squamous epithelial cells of normal tonsil tissue and normal esophageal tissue (indicated by arrows); they have brown cytoplasm-positive localization in the tumor cells of lung squamous cell carcinoma tumor tissue (indicated by arrows), and they are negative in the tumor cells of lung adenocarcinoma tumor tissue (indicated by arrows).

[0047] The results showed that the antibody can distinguish between lung squamous cell carcinoma and lung adenocarcinoma in the diagnosis of lung cancer, and the antibody expression spectrum meets the theoretical and practical immunohistochemical application standards.

[0048] Example 4 Application of anti-CK5&6 monoclonal antibodies in Western blot (1) Select A431 cell lysate and perform polyacrylamide gel electrophoresis. The loading amount of A431 lysate is 20 μg / lane. The electrophoresis conditions are: 80V, 40min; 120V, 80min.

[0049] (2) Transfer: Remove the gel from the glass plate after electrophoresis in step (1), soak it in transfer buffer for 30 min, and then perform the transfer experiment. The transfer conditions are: 0.4A constant current transfer for 20 min.

[0050] (3) After the transfer is completed, remove the PVDF membrane, rinse the PVDF membrane with ddH2O for 5 minutes, add 10mL of Ponceau red for staining, wash the membrane with methanol for 5 minutes, dry it and set aside. At this time, the red area of ​​the cell protein lysate on the PVDF membrane indicates that the protein lysate on the cells has been transferred to the PVDF.

[0051] (4) Blocking: Take out the PVDF membrane prepared in step (3), activate it with methanol for 30 seconds, and then wash the membrane with ddH2O for 5 minutes. Then add 10 mL of 5% skim milk powder to block the non-specific binding sites on the membrane, and incubate at 60 rpm for 1 hour at room temperature.

[0052] (5) Primary antibody incubation: After step (4), add anti-CK5&6 monoclonal antibodies at dilutions of 1:500, 1:1000, 1:2000, and 1:5000, and incubate at room temperature and 60 rpm for 1 h.

[0053] (6) Secondary antibody incubation: After step (5), add 1:4000 diluted HRP-conjugated goat anti-rabbit IgG polyclonal antibody and incubate at room temperature at 60 rpm for 1 h.

[0054] (7) Development and photography: After step (6), add the prepared ECL developer to the membrane. Place the PVDF membrane in a UV exposure instrument for photography.

[0055] (8) See the image after inversion for details. Figure 3 .

[0056] From the results in the figure, we can see that the anti-CK5&6 rabbit monoclonal antibody can recognize the CK5&6 protein in the A431 cell lysate, with an apparent molecular weight of 60kD. There are target bands at different dilutions and no obvious miscellaneous bands appear, which is consistent with the theory and literature reports. This shows that the antibody can be used for Western blot experimental applications.

[0057] Example 5 Application of anti-CK5&6 monoclonal antibodies in flow cytometry (1) Use cultured A431 cells, wash with PBS and adjust the cell density to 1 M / mL.

[0058] (2) Divide the 1M / mL cells into 3 tubes, 1 tube for negative control experimental cells, 1 tube for isotype control experimental cells, and 1 tube for anti-CK5&6 antibody experimental cells.

[0059] (3) Cell fixation: Add 1 mL of 4% paraformaldehyde fixative to each of the three tubes of cells and fix the cells for 30 min. Then add 1 mL of 1% BSA in PBS and centrifuge at 1400 rpm at room temperature for 5 min.

[0060] (4) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 min.

[0061] (5) Cell membrane disruption: Add 1 mL of 0.1% Triton X-100 permeabilization agent to each of the three tubes of cells and permeabilize the cells for 10 minutes. Then add 1 mL of 1% BSA in PBS and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0062] (6) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 min.

[0063] (7) Primary antibody incubation: Add 100µL of 1% BSA in PBS to one tube of cells, 100µL of 1µg / mL isotype control CK10 antibody to another tube, and 100µL of 1µg / mL anti-CK5&6 rabbit monoclonal antibody to the last tube. Resuspend the cells and incubate at room temperature for 60 minutes. After incubation, add 1mL of 1% BSA in PBS to each tube and centrifuge at 1400rpm at room temperature for 5 minutes.

[0064] (8) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 min.

[0065] (9) Secondary antibody incubation: Add 100 µL of 1:2000 diluted goat Anti-Rabbit IgG H&L (Alexa Fluor®488) to each tube, resuspend the cells and incubate at room temperature for 60 min. After incubation, add 1 mL of 1% BSA in PBS to each tube and centrifuge at 1400 rpm at room temperature for 5 min.

[0066] (10) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 min.

[0067] (11) Flow cytometry detection: Using the isotype control well as the standard, adjust the voltage for flow cytometry detection. The detection results are as follows: Figure 4 shown.

[0068] The results showed that when anti-CK5&6 rabbit monoclonal antibody was used to perform flow cytometry experiments on A431 wild-type tumor cell lines, the isotype control antibody did not recognize the CK5&6 protein on the A431 wild-type cell line (left curve), while the anti-CK5&6 rabbit monoclonal antibody could recognize the CK5&6 protein on the A431 wild-type cell line (right curve). This antibody can be used for flow cytometry detection applications.

[0069] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. An anti-CK5&6 antibody, Features: It includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region CDR-H1 is shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 6; the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

9.

2. The antibody according to claim 1, Features: The antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:2, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

3.

3. The antibody according to claim 1 or 2, Features: The antibody is a rabbit anti-human monoclonal antibody, and the rabbit immunogen comprises the amino acid sequence shown in SEQ ID NO:

1.

4. An expression vector, Features: Contains a nucleic acid encoding the antibody according to claim 1 or 2.

5. A host cell, Features: Comprising the expression vector according to claim 4.

6. Use of the antibody according to any one of claims 1 to 3 in the preparation of an immunoassay tool for detecting and identifying CK5&6 proteins.

7. The use according to claim 6, Features: The immunoassay tool includes reagents, test kits, chips or test strips.

8. The use according to claim 6, Features: The immunodetection tool is used for at least one of immunohistochemistry, flow cytometry, and immunoblotting.

9. Use of the antibody according to any one of claims 1 to 3 in the preparation of a diagnostic kit for lung squamous cell carcinoma and lung adenocarcinoma.

10. A kit for detecting and identifying CK5&6 proteins, It is characterized in that Comprising the antibody of claim 3.

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