CN112390895B

CN112390895B - CK20 antigen, hybridoma cell strain, monoclonal antibody and application thereof

Info

Publication number: CN112390895B
Application number: CN202011462032.5A
Authority: CN
Inventors: 王亚杰; 徐星星; 柴素真; 张东辉; 常秋霜; 翟晋豫; 刘文弟; 刘畅; 李三华; 牛银银; 齐华
Original assignee: Henan Celnovtebio Biotechnology Inc
Current assignee: Henan Celnovtebio Biotechnology Inc
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2023-06-20
Anticipated expiration: 2040-12-11
Also published as: CN112390895A

Abstract

The invention relates to CK20 antigen, hybridoma cell strain, monoclonal antibody and application thereof. The invention combines the characteristics of cytokeratin sequences, selects the head and tail sequences of CK20 hypermutation, adopts a connecting element to connect the head and tail sequences, carries out gene synthesis according to the sequence of the head, the connecting element and the tail, inserts the gene synthesis into a prokaryotic expression vector, and expresses and purifies the gene to obtain CK20 antigen; taking the cell as an immunogen to immunize a BALB/C mouse, taking spleen cells of the mouse and SP2/0 cells to fuse, obtaining monoclonal cells by a limiting dilution method, screening positive hybridoma cells by an indirect ELISA method, and finally obtaining hybridoma cell strain C7C11 capable of stably secreting mouse anti-human CK20 monoclonal antibodies, wherein the preservation number is CCTCC NO: c2020201; monoclonal antibodies were prepared by in vivo induction in animals, and their specificity and sensitivity were verified by Western Blot and Immunohistochemistry (IHC), respectively. The CK20 monoclonal antibody provided by the invention has better specificity and sensitivity, and can be used for preparing a CK20 in-vitro immunodetection reagent or kit.

Description

CK20 antigen, hybridoma cell strain, monoclonal antibody and application thereof

Technical Field

The invention relates to a CK20 antigen, a hybridoma cell strain, a monoclonal antibody and application thereof, and belongs to the technical field of immunology.

Background

Cytokeratin is a component of intermediate fiber, which consists of a rod-like region of approximately 310 amino acids in the middle, with the N-terminal and C-terminal tails of hypervariability at both ends. Due to the specific composition of the intermediate fiber, its specific structure is formed: the amino acid sequence of the stem part has higher homology, the stem part of the same type of intermediate fiber has homology of 70-90%, however, cytokeratin is an important component of the intermediate fiber, 54 members are found at present, so that low specificity caused by homology easily occurs when an immunological experiment is carried out, and the specific recognition of a certain cytokeratin is particularly important.

Cytokeratin 20 (CK 20) as an important component of cytokeratin, consists of 424 amino acids and has a relative molecular weight of 48000. In normal tissues, CK20 is expressed in intestinal mucosal cells, gastric mucosal cells, pyloric gland cells, duodenal mucosal cells, urinary umbrella cells, merkel cells. CK20 is often expressed in tumor tissues in colorectal cancer, cholangiocarcinoma, pancreatic ductal adenocarcinoma, mucinous ovarian tumor, merkel cell tumor, and transitional cell carcinoma. It is reported that CK20 is expressed in a significant difference in different cancers among tumors. Whereas tumor cells expressing CK20 are derived from normal epithelial cells that themselves express CK 20. Thus, CK20 is commonly used to identify the tissue source of a tumor.

The commonly used in vitro immune detection method of CK20 is an Immunohistochemical (IHC) method, and an anti-human CK20 monoclonal antibody plays a key role in the process of detecting the expression condition of CK20 in isolated tissue cells by the IHC method, and the sensitivity and the specificity of the detection method are determined by the capability of the anti-human CK20 monoclonal antibody to specifically bind to human CK20 protein. The existing CK20 in-vitro immunodetection antibody is mostly imported, has few types, has sensitivity to be improved and is high in price. The CK20 monoclonal antibody prepared at present is mostly immunized by natural extraction, prokaryotic expression of CK20 full-length recombinant protein or artificial synthetic polypeptide. However, because the amino acid sequence of the middle fiber stem part has higher homology, the problem of low specificity caused by homology easily occurs when the CK20 full-length recombinant protein is used as an immunogen to prepare an antibody, so that the screening difficulty of monoclonal cell strains is increased. The use of full-length CK20 protein for antibody production in the development of diagnostic reagents makes it easy to obtain low-specificity antibodies that recognize various cytokeratins, resulting in failure of development. Furthermore, the use of synthetic polypeptides as immunogens allows for the determination of antibody recognition epitopes, but the differences in the three-dimensional structure of the protein and the interactions between different molecules of the polypeptide fragments at different positions may result in differences that affect the judgment and diagnosis.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a CK20 antigen, which is a fusion protein obtained by adding a connecting element between the head and tail fragments of human CK20 protein and can be used for immunizing animals to obtain CK20 antibodies.

Next, the present invention provides a murine anti-human CK20 monoclonal antibody prepared from the above antigen as an immunogen.

Meanwhile, the invention provides a hybridoma cell strain capable of stably secreting the mouse anti-human CK20 monoclonal antibody and a method for preparing the CK20 monoclonal antibody by using the hybridoma cell strain.

The invention further provides application of the mouse anti-human CK20 monoclonal antibody in preparation of a CK20 in-vitro immunodetection reagent or a kit.

Finally, the invention provides a CK20 immunohistochemical detection kit.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the first aspect of the present invention provides a CK20 antigen, which is a fusion protein obtained by adding a connecting element between the head and tail fragments of a human CK20 protein, and specifically comprises the following structure:

1) First sequence: the head of the human CK20 protein has an amino acid sequence shown in SEQ ID NO:1 is shown in the specification;

2) A connecting element: (GGGGS) n, n=3, amino acid sequence as set forth in SEQ ID NO:5 is shown in the figure;

3) Second sequence: the tail part of the human CK20 protein has an amino acid sequence shown in SEQ ID NO:3 is shown in the figure;

the above CK20 antigen can be used as an immunogen to prepare an anti-human CK20 antibody.

The second aspect of the present invention provides a method for producing the above CK20 antigen: the head nucleotide sequence of CK20 SEQ ID NO:2 and the tail nucleotide sequence SEQ ID NO:4, a nucleic acid sequence of the connecting element SEQ ID NO:6, cloning the recombinant strain into a pET-28a expression vector after artificial synthesis, constructing a pET-28a-CK20-Linker expression vector, and transforming escherichia coli to express and purify to obtain the CK20 antigen.

In a third aspect, the present invention provides a murine anti-human CK20 antibody, which is a monoclonal antibody prepared from the above CK20 antigen as an immunogen. Immunizing a female BalB/c mouse with the age of 6-8 weeks by adopting the expressed CK20 fusion protein, fusing spleen cells of the mouse with SP2/0 cells to obtain monoclonal cells by a limiting dilution method, and screening positive hybridoma cells by an indirect ELISA method to obtain a hybridoma cell strain capable of secreting anti-CK 20 specific antibodies.

The fourth aspect of the invention provides a mouse anti-human CK20 monoclonal antibody hybridoma cell strain, which is: C7C11, accession number: cctccc NO: C2020201. the CK20 antigen is adopted to immunize a female BALB/c mouse with the age of 6-8 weeks, spleen cells of the mouse are fused with SP2/0 cells, monoclonal cells are obtained through a limiting dilution method, positive hybridoma cells are screened through an indirect ELISA method, and a hybridoma cell strain capable of stably secreting anti-CK 20 specific antibodies is obtained.

The fifth aspect of the present invention provides a method for preparing a mouse anti-human CK20 monoclonal antibody, comprising inoculating the above anti-human CK20 monoclonal antibody hybridoma cell strain into the abdominal cavity of a mouse, collecting ascites, and purifying by Protein a column to obtain the CK20 monoclonal antibody.

The mouse anti-human CK20 monoclonal antibody can be used for preparing a CK20 in-vitro immunodetection reagent or a kit.

The fifth aspect of the present invention provides a CK20 immunohistochemical detection kit, comprising the above mouse anti-human CK20 monoclonal antibody, useful for in vitro immunodetection of CK20 expression in tissue cells.

The invention has the beneficial effects that:

1. the CK20 antigen provided by the invention is a fusion protein obtained by adding a connecting element between the head and tail fragments of human CK20 protein. The head and tail segments of the high variation are selected, the relatively conservative rod sequence is replaced by a linker, and the linker is used as an antigen, so that the low-specificity immune effect easily generated when the CK20 full-length recombinant protein is used as an immunogen is avoided, and the immune hit rate to the CK20 is improved; meanwhile, the addition of the connecting element is beneficial to the full exposure of the structures of the head protein and the tail protein and the mutual influence of the structures of the head protein and the tail protein, and the method contains more epitopes compared with the synthetic polypeptide; and the linker has weak immunogenicity and is not easy to generate antibodies, thereby avoiding the generation of nonspecific antibodies.

2. The invention provides a hybridoma cell strain C7C11 capable of stably secreting a mouse anti-human CK20 monoclonal antibody, which has the preservation number of: cctccc NO: c2020201, date of preservation: 10/28 of 2020, deposit unit: china center for type culture collection, preservation address: the Chinese university of Wuhan is prepared by taking a fusion protein obtained by adding a connecting element between head and tail fragments of human CK20 as an antigen to immunize a BalB/c mouse through an immunological method; the hybridoma cell strain can secrete the mouse anti-human CK20 monoclonal antibody with strong specificity and high sensitivity, and has high expression quantity and good expression stability.

3. The mouse anti-human CK20 monoclonal antibody provided by the invention can be used for detecting CK20 expression condition of isolated tissue cells by in vitro immunity, has the characteristics of strong specificity, high sensitivity and good accuracy, and compared with an expensive imported antibody, the mouse anti-human CK20 monoclonal antibody provided by the invention has the advantages of stronger specificity and low price compared with an imported antibody, and can reduce patient burden and diagnosis cost.

4. The preparation method of CK20 antigen can avoid nonspecific antibody generated by cytokeratin homology rod-shaped region, and provides a referential method for antigen preparation in cytokeratin series monoclonal antibody preparation.

Drawings

FIG. 1 shows the purification result of CK20 recombinant protein in the invention, wherein M is Marker, and a lane 1 sample is purified recombinant protein;

FIG. 2 is a diagram showing the result of Westernblot for preparing CK20 monoclonal antibody of the invention, M is Marker, lane 1 is A549 cell lysate, lane 2 is Hela cell lysate, and lane 3 is mouse small intestine tissue lysate;

FIG. 3 is a graph showing the results of immunohistochemical detection of murine anti-human CK20 monoclonal antibody and Ks20.8 prepared in accordance with the present invention, wherein the tissue examined in the graph is the appendix;

FIG. 4 is a graph showing the results of immunohistochemical detection of the murine anti-human CK20 monoclonal antibody and Ks20.8 prepared in accordance with the present invention, wherein the tissue to be detected is intestinal tissue;

FIG. 5 is a graph showing the results of immunohistochemical detection of murine anti-human CK20 monoclonal antibody and Ks20.8 prepared in the present invention, wherein the tissue to be detected is urothelial tissue.

Detailed Description

The invention is further described in connection with the following detailed description, but the scope of the invention is not limited thereto:

example 1

The CK20 antigen in this example is a fusion protein obtained by adding a linking element between the head and tail fragments of human CK20 protein.

The Uniprot accession number of human CK20 is P35900, which contains 424 amino acids, wherein 1-69 are the head, 70-377 are the stem, 378-424 are the tail, and the amino acid and nucleotide sequences of the head are shown in SEQ ID NO:1 and SEQ ID NO:2, the tail amino acid and nucleotide sequence is shown in SEQ ID NO:3 and SEQ ID NO:4. the head and tail sequences were chosen as the first and second sequences, respectively, according to the CK20 sequence and structure published by Uniprot.

The elastic connecting element (linker) commonly used at present is (GGGGS) n, and the elastic connecting element does not influence the conformation and the function of proteins at two ends. In order to ensure the distance between the head and the tail of cytokeratin, reduce the steric hindrance of the head and the tail, play the role of the head and the tail, ensure that the length of a linker is more than 3.5nm, and n is at least equal to 2, namely the linker has at least 10 amino acids, the invention selects n=3, namely (GGGGGGS) ₃ . The amino acid and nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6.

in summary, the CK20 antigen consists of the following structure:

3) Second sequence: the tail part of the human CK20 protein has an amino acid sequence shown in SEQ ID NO: 3.

Example 2

The CK20 antigen in this example is prepared by combining the head nucleotide sequence of CK20 with SEQ ID NO:2 and the tail nucleotide sequence SEQ ID NO:4, a nucleic acid sequence of the connecting element SEQ ID NO:6, cloning the obtained product into a pET-28a expression vector after artificial synthesis, constructing a pET-28a-CK20-Linker expression vector, and transforming escherichia coli to express and purify the obtained product.

1) Adding a Linker nucleic acid sequence in the middle of the head and tail nucleic acid sequences of CK20 for artificial synthesis, introducing enzyme cutting sites NdeI and XhoI at two ends of the sequence, cloning into a pET-28a expression vector after synthesis, and constructing the pET-28a-CK20-Linker expression vector.

2) In the competent of the recombinant plasmid transformed BL21 (DE 3) escherichia coli constructed in the above way, single colonies are selected and cultured in 4mL of LB liquid medium containing kanamycin (50 mug/mL), and IPTG with the final concentration of 1mM is added for expression analysis, so that the strain capable of expressing the target protein is preserved.

3) Can be as described aboveThe strain expressing the target protein is inoculated in 350mL LB liquid medium containing kanamycin (50 mu g/mL) and cultured at 37 ℃ until OD ₆₀₀ Reaching 0.8, IPTG was added at a final concentration of 0.4mM, and expression was induced at 37℃for 4 hours.

4) The above-mentioned cells inducing expression were collected by centrifugation, resuspended in ice-cooled 10mM PBS, sonicated (350W, 20min, working 3s, resting 3 s), centrifuged at 12000g for 10min, and the supernatant solution was collected for the next purification.

5) The supernatant was filtered through a 0.45 μm filter, and the filtrate was subjected to nickel agarose gel purification. The filtrate was loaded into the equilibrated gel and the flow rate was controlled at 1mL/min. Washing with 10mM PBS to remove unadsorbed protein, linear eluting with PBS and PBS containing 0.5M imidazole, collecting different elution peaks, performing SDS-PAGE to identify protein, ultrafiltering to remove more than 90% purity sample to 10mM PBS to obtain CK20 fusion protein antigen, and purifying recombinant protein as shown in figure 1.

Example 3

The hybridoma cell line C7C11 in this example was prepared by a method comprising the steps of:

1) Animal immunization: mixing the purified CK20-Linker protein with an equal volume of Freund's complete adjuvant, immunizing a BalB/c mouse with the age of 6-8 weeks by adopting a subcutaneous injection method, wherein the immunization dose is 100 mug/mouse, performing second immunization after two weeks, emulsifying an antigen by adopting Freund's incomplete adjuvant, and adopting an intraperitoneal injection method with the immunization dose the same as that of the first immunization dose. And taking tail blood after the second immunization, carrying out gradient dilution by an indirect ELISA method to determine the serum titer, and selecting a mouse with the highest antibody titer for tail vein impact immunization, wherein the impact dose is 50 mug/mouse. .

2) Cell fusion: myeloma cells adopt BalB/c sp2/0 and are in logarithmic growth phase during fusion; aseptically picking spleen of the mice subjected to the impact immunization to prepare spleen cell single-cell suspension; the spleen cells of the mice and myeloma cells are mixed at a ratio of 1:5, 50% PEG-1500 (PH 8.0) at 37 ℃ is dripped into the mixture, the mixture is incubated for 90 seconds at room temperature, an incomplete culture medium is added into the mixture to carry out termination reaction, HAT culture medium is added into the mixture to be suspended and mixed evenly after supernatant is removed by centrifugation, the volume of the mixture is fixed to 150mL, the mixture is dripped into a 96-well cell culture plate, and the mixture is placed into a 5% CO2 constant temperature incubator at 37 ℃ for culture for 7 days.

3) Screening and cloning: selecting cell clone 7-10 days after fusion, screening, detecting (ELISA, IHC), selecting cell hole with high ELISA positive value and IHC reactivity as positive hole, limiting dilution of positive hole cell, measuring ELISA value 5-6 days after each limiting dilution, selecting OD ₄₅₀ Limiting dilution is carried out on the monoclonal hole with higher positive value until the result of ELISA measurement of the whole plate of the 96-well plate is positive, and then the cell of the hole is determined to be a monoclonal cell strain.

4) The specific experimental results are as follows: the fusion cells were plated in a total of 8 plates, 750 wells. The positive wells 237 were co-screened for 31.6% positive rate by ELISA. ELISA detection antigen is recombinant expressed CK20 protein, the antigen coating amount is 50 ng/hole, and the secondary antibody dilution ratio is 1:5000, reaction process: coating at 37 ℃ for 2 hours, sealing at 37 ℃ for 2 hours, incubating a primary antibody at 37 ℃ for 30 minutes, incubating a secondary antibody at 37 ℃ for 30 minutes, adding TMB for color development, reading, and selecting a hole with a value/negative value of more than 2.1 as positive, wherein the value of the negative hole is 0.08. Based on ELISA, removing cell strains with low background, nonspecific staining and affinity by IHC detection, screening out 3 strains for IHC detection, and selecting cell strain C7C11 with high specificity and affinity for experiment.

The obtained C7C11 hybridoma cell strain is frozen for preservation, and the preservation number is as follows: cctccc NO: c2020201, date of preservation: 10/28 of 2020, deposit unit: china center for type culture collection, preservation address: chinese university of Wuhan.

Example 4

The mouse anti-human CK20 monoclonal antibody in the embodiment is prepared by adopting a mouse abdominal cavity induction method, and comprises the following steps:

BalB/c mice of 8-10 weeks of age were intraperitoneally injected with 0.5ml liquid paraffin, after one week each mouse was intraperitoneally injected with 1ml syringe with 5X 10 cells as a suspension of monoclonal cells which were resuspended by washing with PBS ⁶ 3 mice were plated per cell line. Collecting ascites after accumulating mouse ascites, centrifuging to obtain supernatant, purifying ascites by Protein A column chromatography, purifying monoclonal antibody by ultraviolet spectrophotometerThe concentration was adjusted to 1.0mg/mL, and the mixture was stored in a sub-package at-20 ℃.

Example 5

In this example, western blotting (Western-Blot) was used to detect the specificity of murine anti-human CK20 monoclonal antibodies.

1. Sample preparation: a549, hela cell line, mouse small intestine tissue homogenate were prepared, lysed with RIPA containing 1mM PMSF, and 5 XSDS-PAGE Loading buffer was added thereto to prepare a sample.

2. Electrophoresis and transfer: each sample was loaded with 30 μg and run at 80V for 20min and then 120V to bromophenol blue to the bottom of the gel. Then starting transferring film, soaking NC film and filter paper in transfer buffer solution in advance, placing the sandwich form in a wet transferring film instrument, and the sequence is as follows: electrode (-) -sponge-filter paper-gel-NC membrane-filter paper-sponge-electrode (+), constant pressure 90v,90min.

3. Closing: NC membranes were immersed in blocking solution (TBST with 5% skim milk) and incubated for 2 hours at 37 ℃.

4. Incubation resistance: NC membrane was placed in a mouse anti-human CK20 monoclonal antibody (antibody diluted 1:2000 with blocking solution), incubated for 1h at 37℃in an incubator, and washed 3 times with TBST for 5min each.

5. Secondary antibody incubation: NC membrane was placed in HRP-labeled goat anti-mouse IgG secondary antibody (antibody diluted 1:5000 in blocking solution), and after incubation for 30min in a shaker at 37℃TBST was washed 3 times for 5min each.

6. Color development: the components A and B in the ECL luminous liquid are as follows 1:1 are added on NC film after being evenly mixed, and are exposed in an exposure instrument.

The experimental results are shown in fig. 2, wherein M is Marker, lane 1 is a549 cell lysate, lane 2 is Hela cell lysate, lane 3 is mouse small intestine tissue lysate, and as can be seen from fig. 2, the anti-human CK20 antibody can react with three samples, the band is clear, no impurity band is generated, and the murine CK20 protein can be identified.

Example 6

The CK20 in-vitro immunoassay kit comprises a primary antibody reagent, a peroxidase blocking solution, a secondary antibody reagent, a DAB display substrate, a DAB chromogenic buffer solution and a hematoxylin staining solution, wherein the primary antibody reagent comprises a mouse anti-human CK20 monoclonal antibody secreted by hybridoma cell strain C7C11, the secondary antibody reagent is an HRP-marked goat anti-mouse/rabbit polymer, and the secondary antibody reagent is purchased from Henan Saint Biotechnology Co.

1. 131 parts of tissue wax block is selected, and comprises abnormal and normal tissues such as lung, breast, stomach, colorectal, appendix, intestine, cervix, skin, esophagus, urothelium, pancreas, brain, thyroid, tonsil, placenta, testis, prostate and the like, and is continuously sliced for 2 pieces by using a Leica tissue slicer, wherein the tissue thickness is 3 mu m, and the tissue is baked for 2 hours at 65 ℃.

2. The antibody of the invention is subjected to immunohistochemical staining test by using a manual immunohistochemical method, and the specific steps are as follows: performing HIGH-temperature repair for 20min by adopting DAKO HIGH pH repair liquid; after cooling, using 100 mu l of endogenous peroxidase sealing liquid to incubate for 5min at room temperature, and soaking the cleaning liquid for 2 times and 5 min/time; the monoclonal antibody of the anti-human CK20 is diluted by an antibody diluent according to a ratio of 1:1000, and a control antibody Ks20.8 is respectively dripped with 100 μl of the antibody, 100 μl of the diluted antibody is dripped, incubated for 30min at 37 ℃, and soaked in a cleaning solution for 2 times and 5 min/time; using 100 μl of enzyme-labeled polymer, incubating at room temperature for 20min, and soaking in the cleaning solution for 2 times and 5 min/time; 100 μl of DAB color development solution is used, incubated for 5min at room temperature, and soaked in purified water for 2 times and 5 min/time; 100 μl hematoxylin counterstain was added dropwise, incubated at room temperature for 5min, and rinsed with tap water to return to blue.

3. Dewatering, transparentizing and sealing: washing with deionized water for 3min;85% ethanol for 1min;95% ethanol for 1min;100% ethanol 1min,2 times; xylene 1min,2 times; and (5) sealing the neutral resin.

4. And (5) microscopic examination: and the detection result is observed under a microscope, the intracellular CK20 protein is localized to cytoplasm, and the tissue is stained clearly.

5. The immunohistochemical results were judged to be negative and positive only, and the summary results are shown in the following table, wherein C7C11 is consistent with Ks20.8 in yin and yang, and no difference in staining results exists.

The anti-CK 20 antibody C7C11 and Ks20.8 recognition proteins are positioned in cytoplasm, the cell positioning is accurate, the staining is clear, and no background exists, so that the C7C11 can accurately recognize the target protein, and the specificity is equivalent to the Ks20.8 recognition capability; in partial tissue sections, the C7C11 staining intensity was stronger than Ks20.8.

Wherein, select 3 positive tissue sections to take a picture, record:

FIG. 3 is a graph showing the results of immunohistochemical detection of the murine anti-human CK20 monoclonal antibodies C7C11 and Ks20.8 prepared in accordance with the present invention, wherein the tissue examined is the appendix;

FIG. 4 is a graph showing the results of immunohistochemical detection of the murine anti-human CK20 monoclonal antibodies C7C11 and Ks20.8 prepared in accordance with the present invention, wherein the tissue to be detected is intestinal tissue;

FIG. 5 is a graph showing the results of immunohistochemical detection of the murine anti-human CK20 monoclonal antibodies C7C11 and Ks20.8 prepared in the present invention, wherein the tissue to be detected is urothelial tissue.

<110> Henan Sainot Biotechnology Co., ltd

<120> CK20 antigen, hybridoma cell line, monoclonal antibody and application thereof

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 69

<212> PRT

<213> Human

<221> CK20 head

<400> 1

Met Asp Phe Ser Arg Arg Ser Phe His Arg Ser Leu Ser Ser Ser Leu

1 5 10 15

Gln Ala Pro Val Val Ser Thr Val Gly Met Gln Arg Leu Gly Thr Thr

20 25 30

Pro Ser Val Tyr Gly Gly Ala Gly Gly Arg Gly Ile Arg Ile Ser Asn

35 40 45

Ser Arg His Thr Val Asn Tyr Gly Ser Asp Leu Thr Gly Gly Gly Asp

50 55 60

Leu Phe Val Gly Asn

65

<210> 2

<211> 207

<212> DNA

<213> Human

<221> CK20 head

<400> 2

atggatttca gtcgcagaag cttccacaga agcctgagct cctccttgca ggcccctgta 60

gtcagtacag tgggcatgca gcgcctcggg acgacaccca gcgtttatgg gggtgctgga 120

ggccggggca tccgcatctc caactccaga cacacggtga actatgggag cgatctcaca 180

ggcggcgggg acctgtttgt tggcaat 207

<210> 3

<211> 47

<212> PRT

<213> Human

<221> CK20 tail

<400> 3

Asp Val Lys Thr Thr Glu Tyr Gln Leu Ser Thr Leu Glu Glu Arg Asp

1 5 10 15

Ile Lys Lys Thr Arg Lys Ile Lys Thr Val Val Gln Glu Val Val Asp

20 25 30

Gly Lys Val Val Ser Ser Glu Val Lys Glu Val Glu Glu Asn Ile

35 40 45

<210> 4

<211> 141

<212> DNA

<213> Human

<221> CK20 tail

<400> 4

gacgtaaaaa ctacagaata tcagttaagc accctggaag agagagatat aaagaaaacc 60

aggaagatta agacagtcgt gcaagaagta gtggatggca aggtcgtgtc atctgaagtc 120

aaagaggtgg aagaaaatat c 141

<210> 5

<211> 15

<212> PRT

<213> artificial sequence

<221> connecting element

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 6

<211> 45

<212> DNA

<213> artificial sequence

<221> connecting element

<400> 6

ggtggtggtg gttccggtgg tggtggttcc ggtggtggtg gttcc 45

Claims

1. A CK20 antigen, which is a fusion protein obtained by adding a connecting element between the head and tail fragments of a human CK20 protein, and specifically comprises the following structure:

2. The method of preparing CK20 antigen according to claim 1, wherein the head nucleotide sequence of CK20 is SEQ ID NO:2 and the tail nucleotide sequence SEQ ID NO:4, a nucleic acid sequence of the connecting element SEQ ID NO:6, cloning the recombinant strain into a pET-28a expression vector after artificial synthesis, constructing a pET-28a-CK20-Linker expression vector, transforming escherichia coli, and carrying out expression purification to obtain the CK20 antigen.

3. Use of the CK20 antigen as claimed in claim 1 as an immunogen in the preparation of anti-CK 20 antibodies.

4. A mouse anti-human CK20 monoclonal antibody hybridoma cell strain, characterized in that the hybridoma cell strain is: C7C11, accession number: cctccc NO: C2020201.

5. a murine anti-human CK20 monoclonal antibody, wherein said monoclonal antibody is secreted by the hybridoma cell line C7C11 of claim 4.

6. The method for preparing the mouse anti-human CK20 monoclonal antibody of claim 5, comprising the following steps: the method for preparing the monoclonal antibody comprises inoculating the mouse anti-human CK20 monoclonal antibody hybridoma cell strain of claim 4 into the abdominal cavity of a mouse, collecting ascites, and purifying to obtain the monoclonal antibody.

7. The use of the murine anti-human CK20 monoclonal antibody of claim 5 in the preparation of a CK20 in vitro immunoassay reagent or kit.

8. A CK20 immunohistochemical detection kit, wherein said kit comprises an anti-agent comprising the murine anti-human CK20 antibody of claim 5.

9. The kit of claim 8, further comprising a peroxidase blocking solution, a secondary antibody reagent, a DAB display substrate, a DAB chromogenic buffer, a hematoxylin staining solution, wherein the secondary antibody reagent is an HRP-labeled goat anti-mouse/rabbit polymer.