Anti-mucin6 protein monoclonal antibody and preparation method and application thereof

Abstract

The present application relates to a kind of monoclonal antibody that can identify human Mucin6 antigen, its preparation method and its use in immunodetection.The above technical solution selects the amino acid of 1950-2050 of Mucin6 protein as antigen peptide, codon optimization becomes the gene fragment suitable for expressing in escherichia coli BL21, and finally the recombinant protein containing Mucin6 protein fragment and histidine tag.The recombinant protein is immunized to mouse, and after cell fusion, screening and subcloning, obtain the mouse hybridoma cell strain 37D10 and the anti-Mucin6 protein monoclonal antibody secreted by the cell strain.The antibody obtained by the present application has high specificity, sensitivity, and can specifically recognize the cell expressing Mucin6 protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, and particularly relates to an anti-Mucin 6 protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] Mucin 6 (MUC6) is a kind of secreted mucin, and its encoding gene is located in the short arm of chromosome 11, 1 region, 5 band, and encodes a protein containing 2439 amino acid residues, which includes a cysteine-rich N-terminal domain, a highly glycosylated domain containing abundant serine, threonine and proline, and a C-terminal cysteine knot, which plays an important role in the formation of MUC6 oligomers. Under physiological conditions, mucin MUC6 is mainly expressed in normal gastric mucosa tissue, and plays a role in lubricating and protecting the gastric mucosa. However, with the deepening of research, it is found that MUC6 is abnormally expressed in many tumors, and plays an important role in the occurrence and development of tumors. In addition to the protein skeleton of mucin, i.e. the mucin core peptide and a large number of sugar chains attached to the core peptide, MUC6 usually also has O-glycan containing α1,4 N-acetylglucosamine terminal residues (α1,4-linked N-acetylglucosamine residues, αGlcNAc). This special structure plays an important role in the process of MUC6 function, and its role in tumor biological behavior has been further elucidated as the importance of MUC6 in tumor pathological process is highlighted. These lay a foundation for further understanding the role and mechanism of MUC6, and provide a research basis and theoretical basis for the prevention, early diagnosis and treatment monitoring of tumors.

[0003] Studies have shown that in gastrointestinal tumors, the expression of MUC6 varies with the type and stage of tumor. In the occurrence and development of gastric cancer, the expression amount of MUC6 protein gradually loses, and the expression of MUC6 may be related to the Lauren classification, differentiation degree, tumor invasion degree and stage of tumor. Studies have shown that the expression of MUC6 in mixed gastric cancer is higher than that in intestinal type and diffuse type gastric cancer, and the expression of MUC6 in mucous adenocarcinoma is higher than that in low differentiation and medium-high differentiation gastric cancer. And the degree of loss of MUC6 is closely related to the poor prognosis of gastric cancer.

[0004] In pancreatic cancer tumor cells, up-regulation of MUC6 expression can inhibit the invasion and migration ability of tumor cells. Moreover, MUC6 can induce the formation of Tn antigens in breast tumor tissue, and the Tn antigens can activate Th17 type immune cells, promote tumor cell apoptosis and inhibit tumor progression. Therefore, MUC6 plays a very important role in inhibiting the occurrence and development of tumors.

[0005] In the pathological diagnosis of gastrointestinal tumors, MUC6 is often used as an important index for immunohistochemical detection. By detecting the expression of MUC6 in tumor tissues, pathologists can assist in tumor typing, staging and prognosis evaluation. For example, in the pathological diagnosis of gastric cancer, the expression of MUC6 can be combined with the expression of other mucins (such as MUC2, MUC5AC) to determine the pathological type and differentiation degree of the tumor. SUMMARY

[0006] The present application provides an anti-Mucin6 protein monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2.

[0007] Further, the monoclonal antibody is a murine monoclonal antibody, and the clone number is 37D10.

[0008] The present application also provides a preparation method of an anti-Mucin6 protein monoclonal antibody, and the antigen for immunizing mice is a recombinant protein, which is expressed by E. coli.

[0009] Further, the recombinant protein comprises a Mucin6 protein fragment and a HIS protein tag.

[0010] Further, the Mucin6 protein fragment is a fragment of 1950-2050 sites, which is the amino acid sequence shown in SEQ ID NO. 3.

[0011] Further, the plasmid vector selected in the recombinant process is pET30a.

[0012] The present application also provides a Mucin6 protein immunodetection reagent, which contains the above-mentioned anti-Mucin6 protein monoclonal antibody as an effective component.

[0013] Further, the immunodetection includes immunohistochemical method, immunoblotting method and enzyme-linked immunoassay.

[0014] Different from the prior art, the beneficial technical effects generated by the application are that: the above technical scheme selects the amino acids at the 1950-2050 site of the Mucin6 protein as an antigen peptide, carries out codon optimization, becomes a gene fragment suitable for expression in Escherichia coli BL21 (DE3), and finally obtains a recombinant protein containing a Mucin6 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing mice, and through cell fusion, screening and subcloning, a mouse hybridoma cell strain and an anti-Mucin6 protein monoclonal antibody are obtained. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the Mucin6 protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a comparison chart of immunohistochemical staining results of gastric adenocarcinoma; the left is the antibody prepared by the application, and the right is a commercially available antibody.

[0016] Figure 2 It is a comparison chart of immunohistochemical staining results of gastric surface epithelium; the left is the antibody prepared by the application, and the right is a commercially available antibody. DETAILED DESCRIPTION

[0017] In order to explain the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved of the present application in detail, the following will be described in detail in combination with the specific embodiments listed and the accompanying drawings. The embodiments described in this paper are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0018] In this paper, the term "embodiment" means that the specific features, structures or characteristics described in combination with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing at various positions in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between other embodiments. In principle, in this application, as long as there is no technical contradiction or conflict, each technical feature mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0019] Unless otherwise defined, the meanings of the technical terms used in this paper are the same as those commonly understood by those skilled in the art to which the present application belongs; the use of related terms in this paper is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0020] In the description of the present application, the phrase "and / or" is a description of the logical relationship between objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " herein generally represents that the associated objects before and after are a "or" logical relationship.

[0021] In the present application, the terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0022] In the present application, the "includes", "contains", "has" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of additional elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0023] As the same understanding as in the "Guidelines for Examination", in the present application, the expressions such as "greater than", "less than", "exceed" are understood as not including the number; the expressions such as "above", "below", "within" are understood as including the number. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly limited.

[0024] Preparation of recombinant Mucin6 protein fragment of Example 1

[0025] I. Gene optimization and synthesis

[0026] According to the protein sequence with accession number NP_002470.2 in the NCBI database, the protein fragment of 1950-2050 amino acids is selected, and the gene fragment suitable for expression in Escherichia coli BL211 (DE3) is directly optimized. In the process of PCR, BamH I and Xho I enzyme cutting sites are added at the 5' and 3' ends of the gene, respectively.

[0027] PCR product was recovered after agarose gel electrophoresis, and the recovered fusion protein gene and plasmid vector pET30a for expression were subjected to BamHI and Xhol enzyme digestion, and then recovered after electrophoresis again, and ligated with T4 DNA ligase. The ligation product was transformed into E. coli competent cells BL211 (DE3), and the clones on the plate were inoculated and subjected to bacterial liquid PCR identification. The clones with positive PCR results were selected for sequencing analysis, and the clones with completely correct sequences were used.

[0028] Different antigens were selected for immunization, which can produce antibodies with different binding properties. The presence of multiple variants caused by variable splicing can ultimately lead to different recognition abilities and patterns of different antibodies to cells expressing antigens. The Mucin6 molecule was analyzed according to the published sequence, and based on its structure, antigenicity, hydrophilic or hydrophobic of constituent amino acids, and secondary structure, a suitable region for soluble expression and good immunogenicity was selected for recombinant expression. The 1950-2050 amino acid residues of Mucin6 were selected for codon optimization, with a molecular weight of about 250 kDa. The Mucin6 protein was obtained by designing the sequence optimized prokaryotic expression gene sequence. The recombinant immunogen is composed of Mucin6 protein fragments with antigenicity and protein tags for purification of recombinant proteins. The protein tag is HIS.

[0029] II. Protein expression and purification

[0030] The single colony culture was transferred to 100 mL LB medium at a ratio of 1:100, and kanamycin was added to a final concentration of 10 μg / mL. The culture was incubated at 37°C with shaking until the OD600 was 0.6-0.8. Then 0.5 mmol / L IPTG was added, and the culture was incubated at 16°C with shaking overnight. The bacteria were collected and sonicated. The recombinant protein has a histidine tag, and nickel column was used for affinity purification of the protein. Elution was performed with 500 mmol / L imidazole, and SDS PAGE separation was performed for detection. The concentration of the recombinant Mucin6 protein with a histidine tag was 0.5 mg / mL, which can be used for animal immunization and antibody screening and identification.

[0031] Example 2 Establishment of hybridoma cell lines

[0032] I. Immunization

[0033] The recombinant protein in Example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week old female ICR mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized by subcutaneous injection of 6 points per mouse at a dose of 20 μg per mouse. The mice were boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg per mouse. Seven days after the third boost, the serum of the mice was tested for the titer of anti-antigen polyclonal antibodies by indirect ELISA (wavelength 450 nm). The mouse with the highest titer was given a boost by tail vein injection of the antigen mixed with normal saline at a dose of 20 μg per mouse.

[0034] II. Cell fusion

[0035] The spleen cell suspension of the immunized mice was prepared aseptically and mixed with mouse myeloma cells sp2 / 0 (ATCC Number CRL-8287) at a ratio of 5:1. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the centrifuge tube was placed in a 37°C water bath. Within 1 min, 1 mL of PEG1500 (Roche) was slowly added while stirring the cells. After standing in warm water for 1 min, 10 mL of serum-free IMDM (Sigma) was added, mixed well, and centrifuged at 1000 rpm for 5 min. After discarding the supernatant, 10 mL of serum (PAA) was carefully added to the cells, which were then blown up, and 5 mL of 10x HAT (Sigma) mixed with thymocytes was added, mixed well. Another 25 mL of semi-solid medium containing 2.1% nitrocellulose (Sigma) was added and mixed well, and then evenly poured into 20 cell culture dishes. The cell culture dishes were placed in a wet box and cultured in a 37°C 5% CO2 incubator.

[0036] III. Cloning and ELISA screening of positive hybridoma cells 37D10

[0037] Seven days after fusion, the size and density of the cell colonies were moderate. Under a dissecting microscope, round, solid, and large colonies were sucked into 96-well culture plates containing medium, and cultured in a 37°C 5% CO2 incubator. After 3 days, the cells occupied about 2 / 3 of the bottom area, and 100 μL of supernatant was taken for ELISA screening with the immunogen and synthetic polypeptide, respectively. The positive clones were completely changed, and 200 μL of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, the second ELISA screening was performed, and the positive clones were transferred to 24-well plates containing medium (containing feeder cells and HT) for culture. Five days later, 100 μL of supernatant was taken for the third ELISA screening, and the positive clones were successively transferred to 6-well plates and cell culture bottles for expansion and cryopreservation.

[0038] I. Preparation of ascites

[0039] Logarithmic growth phase cells were washed with serum-free medium and suspended, and about 5 x 10 5 , 1 mL. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites was collected 7 days later. The removed ascites was centrifuged at 4000 rpm for 10 min at 4°C. The middle ascites was carefully sucked out and collected in a centrifuge tube, and stored at 4°C or -20°C.

[0040] II. Purification of the monoclonal antibody

[0041] The antibody was purified from the ascites by affinity chromatography with HiTrap rProtein A FF (GE) according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by the Bradford method. The purified antibody was stored at -20°C.

[0042] Example 4 Identification of the characteristics of the monoclonal antibody

[0043] I. Subclass identification

[0044] Sheep anti-mouse IgG (Beijing Zhongshanjinqiao Biotechnology Co., Ltd.) was diluted to 0.5 μg / mL with 100 mM PBS (pH 7.4), 100 μL was added to each well, and incubated at 4°C overnight. The liquid was poured out, and washed 3 times with PBS containing 0.05% Tween (PBS-T), 200 μL of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37°C for 1 h. The liquid was poured out, and washed 3 times with PBS-T. 0.1 mL of HRP-labeled sheep anti-mouse (κ, λ) antibody or 1:2000 diluted HRP-labeled sheep anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech) was added to the appropriate wells, respectively, and incubated at 37°C for 1 h. The liquid was poured out, and washed 3 times with PBS-T. 50 μL of citric acid buffer (pH 4.0) containing 0.15% ABTS (Southern Biotech) and 0.03% H2O2 was added to each well for color development, and the OD value at 405 nm was determined within 10-20 min.

[0045] The results show that the monoclonal antibody of the present application is an IgG1 type mouse monoclonal antibody.

[0046] II. Determination of affinity constant

[0047] The Mucin 6 recombinant protein prepared in Example 3 was coated, the coating concentration was 2 μg / mL, 100 μL / well, and the coating was performed at 4°C overnight, and PBS-T was washed for 3 times. 200 μL blocking solution was added to each well, and the blocking was performed at 37°C for 2 h, and PBS-T was washed for 3 times. The monoclonal antibody purified in Example 3 was diluted in a gradient of 2 times from 1:200, and finally 1 well was left as a blank control, and the incubation was performed at 37°C for 1 h, and PBS-T was washed for 3 times. HRP-labeled goat anti-mouse secondary antibody was diluted at 1:20,000, 100 μL was added to each well, and the incubation was performed at 37°C for 1 h, and PBS-T was washed for 3 times. 100 μL of citric acid-phosphoric acid buffer containing 0.1% TMB (Sigma) and 0.03% H2O2 was added to each well, and the color development was performed for 10 min, and 50 μL of 0.5 M sulfuric acid solution was added to terminate the reaction. The absorbance value at a wavelength of 450 nm was determined by using an enzyme-labeled instrument. The curve of the OD value corresponding to the dilution multiple of the antibody was drawn, the dilution multiple A corresponding to half of the maximum binding OD value was found, and the affinity constant of the antibody was calculated to be 1.92×10 9 .

[0048]

[0049] III. Specificity of monoclonal antibody reaction and application effect

[0050] The Mucin 6 recombinant protein prepared in Example 1 was taken, and the recognition specificity of the monoclonal antibody of the application was detected by using the immunoblotting method, and 12% polyacrylamide gel electrophoresis was performed. The gel protein band was transferred to a PVDF membrane (Millipore) in a Bio-Rad electrotransfer system according to a conventional method. The membrane was placed in a TBS-T blocking solution containing 5% skim milk at 4°C overnight. The Mucin 6 protein monoclonal antibody purified in Example 3 (1:1000 dilution) was added, and the incubation was performed at 4°C overnight. After the membrane was washed with TBS-T, goat anti-mouse secondary antibody (Beijing Zhongshen Jinqiao Biotechnology Co., Ltd.) diluted at 1:5000 was added, and the incubation was performed at room temperature for 1 h. The membrane was washed with TBST again, ECL ultra-sensitive developing solution (Beijing Puli Lei Gene Technology Co., Ltd.) was added, and the acquisition of the chemiluminescence image data was performed by using a ChemiDoc MP multi-color fluorescence imaging system (Bio-Rad).

[0051] Example 5: Determination of the variable region sequence of the antibody

[0052] Fresh hybridoma cells were taken, and the supernatant was taken to verify the antigen binding property, and it was confirmed that the cell strain for cloning could indeed secrete the required antibody. After the result was confirmed, the 10 6The hybridoma cells were used. Total RNA of the hybridoma cells was extracted by Trizol method. 9 μL of total RNA, 2.5 μL of oligo(dT)12-18 primer (10 mM), and 5 μL of dNTPs were mixed, and then incubated at 70 °C for 5 min and on ice for 5 min, or were denatured according to the reverse transcriptase used. Then, 5 μL of RT buffer (5x), 2.5 μL of DTT (0.1 M), and 1 μL of reverse transcriptase were added, and the mixture was incubated at 42 °C for 1 h. The reaction was terminated by incubation at 70 °C for 15 min, and the obtained cDNA was stored at -20 °C. The obtained first-strand cDNA was subjected to PCR amplification. In a 50 μL reaction system, 25 pmol of each primer was added, and the sequences of the primers for amplification of the heavy chain variable region and the light chain variable region were designed and synthesized according to the mouse monoclonal antibody primer sequences described in the book "Recombinant Antibody" (Science Press, published in 2005) edited by Shen Bei-fen.

[0053] The remaining dNTPs and buffers were added according to the conventional method, and finally, 1 μL of cDNA template and 1 U of hot-start Taq DNA polymerase were added. The PCR amplification program was set as 94 °C for 40 s, 52 °C for 40 s, and 72 °C for 40 s, and was performed for 20 to 25 cycles, and finally, 72 °C was extended for 3 min. The product was stored at 4 °C or was directly subjected to electrophoresis. 20 μL of the PCR product was subjected to electrophoresis analysis, and was separated on a 1.5% agarose gel. The length of the light chain (κ light chain) was between 320-340 bp, and the length of the heavy chain was between 340-370 bp. When the specific product in the region was obtained, the gel was cut and recovered, and was cloned into a T vector or an expression vector for sequencing.

[0054] Example 6. Immunohistochemical tissue chip staining and identification

[0055] I. Chip preparation process

[0056] Each sample was first subjected to HE section staining to determine the tumor site. The full-automatic tissue chip instrument of 3DHISTECH Company was used to prepare the tissue chip. The prepared tissue chip wax block was again placed into a wax block preparation mold, and was placed into a 68 °C oven for 10 min, so that the tissue chip and the wax of the receptor wax block were integrated, and then the mold was gently taken out of the oven, and the semi-melted paraffin was cooled at room temperature for about 30 min. Then, the tissue chip wax block was taken out of the mold, and was cut or stored in a 4 °C refrigerator for standby. After trimming, continuous sections were cut with a thickness of 3 μm, and were floated in 40% alcohol to naturally expand. Then, the separated sections were transferred to warm water at 50 °C for 30 s, and were attached to the sections with a polylysine-treated glass slide. The prepared tissue chip was placed into a 68 °C oven for 2 h for sectioning, and was taken out, cooled at room temperature, and stored in a -4 °C refrigerator.

[0057] II. IHC staining and analysis

[0058] Routine dewaxing of xylene 3 times, 6 minutes each, hydration in 100%, 100%, 95%, 85% gradient ethanol, 3 minutes each, and finally tap water rinse. Antigen retrieval was performed, then the sections were placed in a wet box and rinsed in PBS 3 x 3 minutes. 3% H2O2 was added and incubated for 10 minutes, rinsed in PBS 3 x 3 minutes. The sections were spun down, the primary antibody was added at the appropriate dilution (the first dilution was designed according to the concentration of the antibody, the dilution ratio of the antibody was designed according to the concentration of the antibody) and incubated at room temperature (25°C) for 1 hour, rinsed in PBS 3 x 3 minutes, the secondary antibody was added and incubated at room temperature for 15-30 minutes, rinsed in PBS 3 x 3 minutes, the PBS was spun off, and fresh DAB developing solution was added for color development for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and PBS was returned to blue for 30 seconds. Sequential dehydration was performed according to the alcohol gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes), and finally xylene clearing for 3 minutes, neutral resin mounting.

[0059] The results of immunohistochemical staining were divided into: positive and negative. Positive expression must be at the site of cell and tissue specific antigen to be considered positive. In the case of clear distribution of tissue staining and accurate cell localization, the staining results were further divided according to the difference in staining intensity, as follows:

[0060] 1. The sample is weakly positive; marked as "+";

[0061] 2. The sample is moderately positive; marked as "++";

[0062] 3. The sample is highly positive; marked as "+++".

[0063] 4. The sample is negative, marked as "-".

[0064] III. Data Statistics

[0065] 1. Tumor tissue chip detection results:

[0066] The antibody Mucin6 (37D10) and the commercially available antibody Mucin6 (MRQ-20) were used to simultaneously detect 55 cases of gastric adenocarcinoma and compare the detection results.

[0067] The immunohistochemical results of Mucin6 were statistically analyzed. The entire test process was designed with double-blind design, and the statistical results are as follows:

[0068]

[0069] The results show that the prepared anti-Mucin6 protein monoclonal antibody has accurate staining positioning, clear staining and no non-specific staining, and a clean background. In the immunohistochemical detection, the positive rate and positive intensity are higher than those of the commercial antibody. It is indicated that the Mucin6 of the application has higher sensitivity, and effectively avoids false negative results.

[0070] Figure 1 The figure of gastric adenocarcinoma immunohistochemical staining results (left: the antibody prepared in the application, right: commercial antibody).

[0071] 2. Normal tissue chip detection results:

[0072] The normal tissue chip includes 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed surgical specimens; each tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cell.

[0073] The antibody (37D10) and the commercial antibody are synchronously detected on the normal tissue chip, and the positive and negative detection results are consistent, which indicates that the specificity of the antibody in the normal tissue is equivalent to that of the commercial antibody.

[0074] Figure 2 The figure of gastric surface epithelial immunohistochemical staining results (left: the antibody prepared in the application, right: commercial antibody).

[0075] Finally, it needs to be explained that although the above-mentioned embodiments have been described in the specification and drawings of the application, the patent protection scope of the application cannot be limited. Any equivalent structure or equivalent flow replacement or modification based on the essential concept of the application, using the content described in the specification and drawings, directly or indirectly, and the technical solutions of the above embodiments applied to other related technical fields, are all included in the patent protection scope of the application.

Claims

1. An anti-Mucin 6 protein monoclonal antibody, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a murine monoclonal antibody.

3. A Mucin 6 protein immunoassay reagent, characterized by, The immunodetection reagent contains the anti-Mucin6 protein monoclonal antibody of claim 1 as an effective component.

4. The immunoassay test reagent of claim 3, wherein, The immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

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