CN113087793B

CN113087793B - anti-CK 14 protein monoclonal antibody, cell strain thereof, preparation method and application

Info

Publication number: CN113087793B
Application number: CN202110517835.4A
Authority: CN
Inventors: 周洪辉; 高惠然; 杨清海; 陈惠玲; 王小亚
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2022-04-12
Anticipated expiration: 2041-05-12
Also published as: CN113087793A

Abstract

The invention relates to a monoclonal antibody capable of recognizing human CK14 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. The technical proposal chemically synthesizes 410-432 amino acid at the C terminal of the CK14 protein as antigen peptide, and obtains immunogen after coupling KLH. The CK14-KLH protein is used for immunizing a mouse, and a mouse hybridoma cell line 2G4 capable of efficiently secreting the anti-CK 14 protein monoclonal antibody and the anti-CK 14 protein monoclonal antibody secreted by the cell line are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing CK14 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

anti-CK 14 protein monoclonal antibody, cell strain thereof, preparation method and application

Technical Field

The invention relates to the field of biomedical engineering, in particular to a monoclonal antibody for resisting CK14 protein, a cell strain, a preparation method and application thereof.

Background

The structural proteins of the cells are divided into keratin, vimentin, intertuscular protein and glial acidic protein, wherein the vimentin, the intertuscular protein and the glial acidic protein are expressed in mesenchymal cells, muscle cells and glial cells, Cytokeratin (CK) is mainly and specifically expressed in human epithelial tissues, is approximately composed of subtype, has the effect of maintaining the stability of epithelial cells or tissue structures, and is also involved in signal transduction, cell proliferation regulation, apoptosis, malignant transformation and various stress reactions of epithelial cells, and the expression of specific cytokeratin types exists in different tumor tissues, so that the expression of cytokeratin is not only related to the etiology of tumors, but also is involved in the generation and development processes of the tumor tissues, and can be applied to diagnosis, typing and prognosis of different tumor tissues.

Cytokeratin 14 (CK 14) is a differentiation-specific protein belonging to the acidic cytokeratin family. The expression of CK14 is limited to basal cells of myoepithelium and keratinized stratified epithelium of various origins, and is one of the proteins constituting the cytoskeleton. The research shows that CK14 is highly expressed in malignant tumors such as lung cancer, breast invasive ductal carcinoma and the like, and the research shows that the high expression of CK14 is related to the process, development and prognosis of esophagus cell cancer and cervical canceration.

Disclosure of Invention

The inventor provides an anti-CK 14 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 CK14 protein.

Further, the monoclonal antibody is mouse IgG₁Subtype monoclonal antibodies.

Furthermore, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20761.

The inventor also provides a preparation method of the anti-CK 14 protein monoclonal antibody, which comprises the steps of selecting an amino acid sequence (458 th to 472 th) shown as SEQ ID NO.1 in CK14 protein, adding a cysteine at the carboxyl terminal of the amino acid sequence, and coupling the amino acid sequence with a carrier protein KLH to serve as immunogen.

The inventor also provides a hybridoma cell strain secreting anti-CK 14 protein molecules, wherein the cell strain is a mouse hybridoma cell strain 2G4, the cell strain is preserved in China general microbiological culture Collection center (CGMCC NO 20761) in 9 and 17 months of 2020, and the accession number is CGMCC NO 20761, and the cell strain is the institute of microbiology of China academy of sciences No.3, North Chen West Lu No.1 Hopkins, the rising area of Beijing.

The inventor also provides the application of the monoclonal antibody in CK14 protein immunoassay.

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

The inventor finally provides a CK14 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 14 protein with the amino acid sequence of the light chain variable region as shown in SEQ ID No.5 as the effective component.

Different from the prior art, the invention has the beneficial technical effects that: according to the technical scheme, amino acids 458 th to 472 th from the C terminal of CK14 protein are selected as antigen peptide, immunogen obtained after coupling KLH is used for immunizing a mouse, and a mouse hybridoma cell line 2G4 capable of efficiently secreting anti-CK 14 protein monoclonal antibody and an anti-CK 14 protein monoclonal antibody secreted by the cell line are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing CK14 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph comparing immunohistochemical staining results for esophageal squamous cell carcinoma; the left part is CK14 secreted by 2G4, and the right part is commercial CK 14.

FIG. 2 is a comparison graph of immunohistochemical staining results of tonsils; the left part is CK14 secreted by 2G4, and the right part is commercial CK 14.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

EXAMPLE 1 preparation of immunogen

First, immunogen preparation

Sequence and secondary structure analysis is carried out according to the protein sequence with the accession number of P02533 in Uniprot, and the molecular weight of the CK14 protein with the total length of 472 amino acids is about 51 kDa. According to the parameters of secondary structure (secondary structure) and Surface Accessibility (Surface Accessibility) of the protein predicted by the online server http:// www.cbs.dtu.dk/services/NetSurfP/and by the analysis of its antigenicity index (Jameson BA, Wolf H. the antigenicity index: a novel homology for predicting antigenic derivatives. Compound application biosci.1988,4(1):181-6.), the amino acid sequence SEQ ID NO.1 at position 458-472 is selected as the antigenic peptide for chemical synthesis and for ease of coupling, a cysteine is added at the carboxyl end of the polypeptide to provide thiol coupling.

Coupling and purification of polypeptide

The equine activated keyhole limpet hemocyanin kit from Thermo Scientific (cat # 77653) was selected and operated according to the protocol provided for the kit. For the polypeptide to be conjugated, the free thiol group in the polypeptide is first detected with Ellman's reagent (Thermo Scientific, cat # 22582): adding 100 mu L Ellman reagent stock solution into a 96-well plate, adding 10 mu L polypeptide solution, measuring the ultraviolet absorption value of the solution by a Nano Drop spectrophotometer at the lambda of 412nm, and performing the next step if the OD value is more than 0.15; the OD value is less than 0.15 and more than 0.05, and the polypeptide is added until the requirement is met; and the OD value is less than 0.05, and the polypeptide is returned to the polypeptide synthesis step for quality control. To begin conjugation, 200. mu.L of deionized water was added to each mcKLH package to make up a 10mg/mL solution of KLH, 2mg of hapten was dissolved in 500. mu.L of Imject EDC coupling buffer, 500. mu.L of polypeptide solution was added to 200. mu.L of carrier protein solution, 1mL of deionized water was added to one package of EDC (10mg) and shaken slowly to dissolve completely, 50. mu.L of polypeptide solution was added to mcKLH polypeptide solution and after 2 hours of reaction, unconjugated crosslinker and salts were removed by desalting column treatment to give CK 14-KLH.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The crosslinked polypeptide of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and ICR mice (purchased from Beijing Wintotonghua laboratory animal technology Co., Ltd.) were immunized and injected ventrally at 6-point per mouse at a dose of 60. mu.g/mouse. The booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co., F5506) at a dose of 30. mu.g/mouse. 7 days after 3 times of booster immunization, the titer of multiple antibodies of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by normal saline, and the dosage is 50 mu g/mouse.

Second, cell fusion

A mouse spleen cell suspension with qualified immunity is prepared aseptically, mixed with mouse myeloma cell sp2/0(ATCC number CRL-8287) at a ratio of 5:1, and centrifuged at 1500rpm for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1ml of PEG1500 (Roche) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, and centrifuged at 1000rpm for 5 min. After discarding the supernatant, 10ml of serum (PAA) was added to the supernatant, the cells were carefully blown up, 5ml of thymocytes mixed with 10XHAT (Sigma) was added, and the mixture was mixed well. Then, 25ml of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and poured into 20 cell culture dishes. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

Cloning and ELISA screening of positive hybridoma cells

The size and density of the clone cell mass are moderate 7 days after fusion, and the round, solid and large clone mass is sucked under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and the culture is carried out in a 5% CO2 incubator at 37 ℃. After 3 days, the cell mass was approximately 2/3 basal areas, and 100. mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely replaced and 200. mu.L of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates previously prepared in medium (containing feeder cells and HT). After five days, 100 μ l of supernatant was subjected to a third ELISA screening, and the positive clones were transferred to 6-well plates and cell culture flasks successively for expanded culture and frozen. EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

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) antibody (Southern Biotech Co.) was diluted at 2000 and added in an amount of 0.1ml per wellIncubate in appropriate wells for 1h at 37 ℃. The liquid was decanted and washed 3 times with PBS-T. Add 50. mu.l of 0.15% ABTS (Southern Biotech) and 0.03% H per well₂O₂The reaction was performed in the citric acid buffer (pH4.0), and the OD value at a wavelength of 405nm was measured within 10 to 20 min.

The results show that the monoclonal antibody of the invention is IgG₁Type murine monoclonal antibodies.

Second, determination of affinity constant

CK14 polypeptide prepared in example 1 was coated at a concentration of 2. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 2h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 3 was purified from 1: 200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: 20000 dilution, 100 μ l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The 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. Drawing a curve of OD value corresponding to the dilution factor of the antibody, finding the dilution factor A corresponding to half of the maximum binding OD value, and calculating the affinity constant of the antibody to be 4.8 × 10 by using the following formula⁸。

Reaction specificity and application effect of monoclonal antibody

The CK14 polypeptide prepared in example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, and 12% polyacrylamide gel electrophoresis was performed. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS-T blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibodies to the antibody secreted by the 2G4 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, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 15-30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds, PBS bluing for 30 seconds. Dehydration was carried out in a gradient of 85% (3 min) -95% (3 min) -100% (3 min) alcohol, finally xylene was transparent for 3 min, and the gel was blocked with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

Data statistics

1. Tumor tissue chip detection results:

the antibody CK14(2G4) and the commercially available antibody CK14(LL002) were tested simultaneously on 30 different squamous carcinoma tumor tissue chips and the results were compared.

The immunohistochemical results of CK14 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 14 protein secreted by the 2G4 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In the positive detection, 3 cases of skin squamous carcinoma and 1 case of esophageal squamous carcinoma exist, the number of staining cells and the staining intensity of CK14 antibody secreted by 2G4 are obviously higher than that of CK14 sold in the market; the CK14 antibody secreted by the 2G4 is higher in sensitivity, and false negative results are effectively avoided.

FIG. 1 is a comparison of immunohistochemical staining results for squamous cell carcinoma of skin (CK 14 secreted from 2G4 on the left and CK14 on the right).

2. Detection results of the normal tissue chip:

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

The antibody (2G4) and the commercial antibody (E3) were detected on the normal tissue chip synchronously, and the negative and positive detection results were consistent, indicating that the specificity of the antibody in the normal tissue was equivalent to that of the commercial antibody.

FIG. 2 is a comparison graph of immunohistochemical staining results of tonsils; the left is CK14 secreted by 2G4, and the right is commercial CK14 (E3).

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.

Sequence listing

<110> Fuzhou mai New Biotechnology development Co., Ltd

<120> anti-CK 14 protein monoclonal antibody, cell strain thereof, preparation method and application

<130> 2021

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 1

Gly Lys Val Val Ser Thr His Glu Gln Val Leu Arg Thr Lys Asn

1 5 10 15

<210> 2

<211> 357

<212> DNA

<213> Artificial sequence (Artificial)

<400> 2

caggtccagc tgcaggagtc agggggagtc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt gactattaca tgtattggat tcgccagact 120

ccggaaaaga ggctggagtg ggtcgcaacc attagtaatg gtggtactta cacccactat 180

ttagacagtg taaaggggcg attcaccatc tccagagaca atgccaagaa caacctgtac 240

ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagaggattt 300

acgacggggg ggacgtttgc ttactggggc caagggactc tggtcactgt ctctgca 357

<210> 3

<211> 336

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

gatatcatga tgacccaatc tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatccagtca gagcattgtg tatagggatg gaaacaccta tttagactgg 120

tatctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc ctgacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc actttttcct 300

tatacgttcg gaggggggac caagttggag ataaga 336

<210> 4

<211> 119

<212> PRT

<213> Artificial sequence (Artificial)

<400> 4

Gln Val Gln Leu Gln Glu Ser Gly Gly Val Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Tyr Trp Ile Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Asn Gly Gly Thr Tyr Thr His Tyr Leu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Phe Thr Thr Gly Gly Thr Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 5

<211> 112

<212> PRT

<213> Artificial sequence (Artificial)

<400> 5

Asp Ile Met Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Arg

20 25 30

Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser Leu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg

100 105 110

Claims

1. The monoclonal antibody for resisting the CK14 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 CK14 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG₁Subtype monoclonal antibodies.

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 20761.

6. A hybridoma cell strain capable of secreting anti-CK 14 protein molecules is a mouse hybridoma cell strain 2G4, is preserved in China general microbiological culture Collection center (CGMCC), and has a preservation number of: CGMCC NO 20761.

7. Use of the monoclonal antibody of any one of claims 1-5 in the preparation of a CK14 protein immunodetection reagent.

8. The use according to claim 7, wherein said immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

9. An immunohistochemical detection reagent for CK14 protein, comprising the anti-CK 14 monoclonal antibody of claim 1 as an active ingredient.