CN114560923A - Tumor marker monoclonal antibody and application thereof - Google Patents
Tumor marker monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention discloses an antigenic polypeptide of CK7 protein and a monoclonal antibody prepared by using the antigenic polypeptide, wherein a heavy chain variable region sequence and a light chain variable region sequence of a monoclonal antibody 1 are shown as SEQ ID NO.1 and SEQ ID NO. 2; the heavy chain variable region sequence and the light chain variable region sequence of the monoclonal antibody 2 are shown as SEQ ID NO.3 and SEQ ID NO. 4. The monoclonal antibody prepared by the invention can be used for detecting CK7 protein, such as immunohistochemistry and ELISA. The monoclonal antibody prepared by the invention has good specificity, accuracy and sensitivity.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and particularly relates to a tumor marker monoclonal antibody and application thereof.
Background
Cytokeratin (CK) is mainly distributed in epithelial cells and is the main skeleton protein in keratinocytes, and the main function of the structural protein is to maintain the integrity and continuity of epithelial tissues. Researches show that Cytokeratin (CK) has extremely high conservation and tissue differentiation specificity and is closely related to the proliferation and differentiation of epithelial cells. More than 20 cytokeratins have been identified. Cytokeratins can be further classified into type I and type II according to their different pH values. The type I cytokeratin is slightly acidic and has small relative molecular mass, and mainly comprises CK 9-CK 20; type II cytokeratins are basic and have large relative molecular mass, and mainly comprise CK 1-CK 8. In different epithelial tissues, the expressed cytokeratin subtypes and different cytokeratins are common tumor immunohistochemical markers, and positive expression is found in epithelial cells and mesothelial cells; cancer, mesothelioma, and the like.
CK7 is a more basic intermediate filament protein in the CK family, with a relative molecular mass of approximately 54kDa and is expressed in the upper gastrointestinal tumors, bile duct carcinoma, pancreatic carcinoma, lung carcinoma, ovary carcinoma, endometrium carcinoma and breast carcinoma; the major markers are glandular and transitional epithelia, ovarian, lung and breast epithelia are CK7 positive, while colon, prostate and gastrointestinal epithelia are CK7 negative, positive in pancreatic, biliary, ovarian serous, endometrioid, transitional cell carcinomas. Is often combined with CK20 to be used for the diagnosis and differential diagnosis of breast cancer, lung adenocarcinoma and gastrointestinal adenocarcinoma, and also can be used for the identification of ovarian cancer (CK7+) and colon cancer (CK 7-). In combination with EMA for the diagnosis of synovial sarcoma. Is mainly used for the research of the tumor of epithelial origin. Therefore, it is necessary to develop a monoclonal antibody for CK7 detection for clinical detection of CK7 protein.
Disclosure of Invention
In order to make up the defects of the prior art, the invention provides a monoclonal antibody of CK7 and a preparation method thereof; the second purpose of the invention is to provide an application of the CK7 monoclonal antibody for detecting CK 7.
Therefore, the invention discloses an antigenic polypeptide of the CK7 protein on one hand, the antigenic polypeptide comprises an antigenic polypeptide 1 and an antigenic polypeptide 2, and the amino acid sequences of the antigenic polypeptide 1 and the antigenic polypeptide 2 are as follows:
antigen polypeptide 1: DVVEDFKNKYEDEINHRTAA;
antigen polypeptide 2: QRAKLEAAIAEAEERGELAL are provided.
On the other hand, the invention also discloses a monoclonal antibody prepared by using the antigen polypeptide, wherein the monoclonal antibody can be specifically bound with CK protein, the monoclonal antibody comprises a monoclonal antibody 1 and a monoclonal antibody 2, and the heavy chain variable region sequence and the light chain variable region sequence of the monoclonal antibody 1 are shown as SEQ ID NO.1 and SEQ ID NO. 2; the heavy chain variable region sequence and the light chain variable region sequence of the monoclonal antibody 2 are shown as SEQ ID NO.3 and SEQ ID NO. 4.
Preferably, the heavy chain constant regions of monoclonal antibody 1 and monoclonal antibody 2 of the present invention are of lgG1 type, and the light chain constant regions are of Kappa type.
In still another aspect, the present invention also discloses a method for preparing the monoclonal antibody, which is characterized in that the method comprises the following steps: 1) designing and preparing CK7 protein antigen polypeptide; 2) preparing hybridoma cells specifically expressing anti-CK 7 protein antigen polypeptide monoclonal antibodies; 3) preparing mouse ascites by using the hybridoma cell prepared in the step 2), and purifying the anti-CK 7 protein antigen polypeptide monoclonal antibody from the ascites.
Preferably, the CK7 protein antigen polypeptide in step 1) of the present invention includes antigen polypeptide 1 and antigen polypeptide 2, and the amino acid sequences of the antigen polypeptide 1 and the antigen polypeptide 2 are:
antigen polypeptide 1: DVVEDFKNKYEDEINHRTAA, respectively;
antigen polypeptide 2: QRAKLEAAIAEAEERGELAL are provided.
On the other hand, the invention also discloses application of the CK7 protein antigen polypeptide in preparing an anti-CK 7 protein monoclonal antibody.
In another aspect, the invention also discloses an application of the monoclonal antibody in preparing a CK7 protein diagnostic reagent.
The antigenic polypeptide of the CK7 protein disclosed by the invention is suitable for preparing a monoclonal antibody of a specific epitope for detecting the clinical CK7 protein. The monoclonal antibody of the anti-CK 7 protein antigen polypeptide can specifically recognize CK7 protein, has good specificity, accuracy and sensitivity, and is suitable for preparing different diagnostic reagents of CK7 protein, such as immunohistochemistry, ELISA and the like.
Drawings
FIG. 1CK7(91aa to 403aa) secondary structure.
FIG. 2 is a SDS-PAGE image of monoclonal antibody 1, monoclonal antibody 2 and M marker, respectively.
FIG. 3 is a test chart for monoclonal antibody specificity (wertern blot) wherein 1 is monoclonal antibody 1 and 2 is monoclonal antibody 2.
FIG. 4 shows the results of immunohistochemistry for monoclonal antibody, where 1 is the result for monoclonal antibody 1 and 2 is the result for control antibody.
FIG. 5 calibration curve for sandwich ELISA assay.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1: preparation of CK7 antigen
CK7(UniProtKB-P08729) was analyzed, the head and tail were removed, and the middle 91aa to 403aa were selected for analysis. The secondary structure of this sequence was resolved and the results are shown in FIG. 1. Designing polypeptide antigen by combining secondary structure, designing and synthesizing 2 segments of antigen polypeptide, wherein the sequences are respectively as follows:
antigen polypeptide 1(81aa to 100 aa): DVVEDFKNKYEDEINHRTAA, respectively;
antigen polypeptide 2(238aa to 257 aa): QRAKLEAAIAEAEERGELAL are provided.
Example 2: preparation of anti-CK 7 monoclonal antibody
The preparation of monoclonal antibodies was carried out using conventional hybridoma cell technology, briefly as follows: BALB/c mice were immunized with the CK7 antigen polypeptide prepared in example 1 as an antigen. After three times of immunization, spleen cells were collected, ground and isolated, fused with myeloma Sp2/0 cell line at a ratio of 9:1 by PEG method, resuspended in complete RPMI 1640 medium containing 20% newborn calf serum of HAT, and then spread evenly in 96-well plates at 37 ℃ and 5% CO in 100. mu.l/well2And (5) culturing. After 15 days, 20% of the calf containing HT is replacedComplete RPMI 1640 medium of bovine serum. The culture supernatant was collected and tested for positive clones by ELISA coated with CK7 antigen. And (3) carrying out cloning culture on the screened cell strains with high antibody titer and good forms by adopting a limiting dilution method until monoclone is obtained, and carrying out amplification culture and storing hybridoma cells. Through the steps, each antigen polypeptide is screened to obtain a hybridoma cell which is respectively named as hybridoma cell 1 (obtained by screening after the antigen polypeptide 1 is immunized) and hybridoma cell 2 (obtained by screening after the antigen polypeptide 2 is immunized).
Example 3: preparation and purification of monoclonal antibody ascites
Taking BALB/c female mice of 8 weeks old, injecting 0.5ml of pristane into the abdominal cavity, and injecting 0.8-1 multiplied by 10 hybridoma cells into each mouse 7 days later6And after 7-10 days, the abdomen of the mouse is obviously enlarged, ascites of the mouse is extracted by an injection needle, the mouse is centrifuged for 10min at 8000rpm at 4 ℃, and the supernatant is collected, namely the monoclonal antibody ascites. The monoclonal antibody is purified by using an octanoic acid-ammonium sulfate precipitation method, 1 volume of ascites is diluted by adding 2 volumes of acetate buffer solution (0.06mol/L, pH value is 4.8), octanoic acid (30 mu L/ml ascites) is added at room temperature while stirring, clarification is carried out at 4 ℃ for 1h, centrifugation is carried out at 12000rpm for 20min, supernatant is collected, immunoglobulin is precipitated by 50% saturated ammonium sulfate, standing is carried out at 4 ℃ for 2h, centrifugation is carried out at 3000rpm for 20min, the precipitate is dissolved by 2 volumes of PBS solution, the purified ascites antibody is obtained after flowing dialysis is carried out at 4 ℃ for 24h, the dialyzed monoclonal antibody is filtered and sterilized by using a 0.22 mu m filter membrane, and subpackaging is carried out, and the volume is 0.1 ml/tube. The monoclonal antibody was subjected to concentration detection using BCA kit (Thermo Scientific), and the detection results of monoclonal antibody 1 (prepared from hybridoma cell 1) and monoclonal antibody 2 (prepared from hybridoma cell 2) were 1.58mg/ml and 1.36mg/ml, respectively. The purified monoclonal antibody was examined by SDS-PAGE, and the results of the examination are shown in FIG. 2, and the purity of the purified monoclonal antibody by SDS-PAGE was 95% or more.
Example 4: detection of monoclonal antibodies
The type and subclass of the monoclonal antibody were identified by using an ELISA kit for identifying the type of the monoclonal antibody from Sigma, and the results showed that the constant regions of the heavy chains and the light chains of the two monoclonal antibodies were of the lgG1 type and of the Kappa type.
See paragraphs [ 0042 ] to [ 0044 ] of CN 113061184A, the affinity constants of the prepared 2 monoclonal antibodies were determined according to this methodology using the purchased CK7 recombinant protein (Shanghai Ye Biotech Co., Ltd.) as the coating antigen, and the results showed that the affinity constants of the 2 monoclonal antibodies prepared by the present invention were 5.0X 109And 5.4X 109。
Total RNA of hybridoma cells was extracted using Trizol reagent. The extracted total RNA was reverse-transcribed into cDNA using oligo (dT) as a primer, and the obtained cDNA was stored at-15 ℃ or lower. Specific nested PCR primers (CN 111393525B) were designed, which were complementary to the first framework region and the constant region of the antibody variable region, and the desired gene was amplified by conventional PCR. The sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1 are shown in SEQ ID NO.1 and SEQ ID NO.2 through sequencing. The heavy chain variable region and the light chain variable region of the monoclonal antibody 2 have the sequences shown in SEQ ID NO.3 and SEQ ID NO. 4. The amino acid sequence information of the variable regions of the two monoclonal antibodies is shown in Table 1.
TABLE 1 monoclonal antibody amino acid sequence information
EXAMPLE 5 use of monoclonal antibodies
5.1 specificity test Using a purchased CK7 recombinant protein (Shanghai leaf Biotech Co., Ltd.) as an antigen, a wertern blot was performed, and a monoclonal antibody was diluted 5000-fold and used as a primary antibody, and an HRP-labeled goat anti-mouse IgG secondary antibody was diluted 10000-fold and used as a secondary antibody, and the results showed that both monoclonal antibodies had good specificity for CK7 protein (FIG. 3).
5.2 immunohistochemical examination referring to example 6 of CN 113061184A, according to the method, two monoclonal antibodies prepared in the research are examined on different tissues, and the results show that the two monoclonal antibodies prepared in the research have good specificity, accuracy and sensitivity on diseased tissues and normal tissues, have no false positive and false negative results (figure 4), and have equivalent or even better effect with a control antibody.
5.3 Sandwich ELISA assays ELISA plates (50ng/0.1 ml/well) were coated with monoclonal antibody 1, monoclonal antibody 2 was used as the detection antibody (labeled with HRP before use, where the HRP label is the conventional sodium periodate method), and the purchased CK7 protein was used as a standard to establish an ELISA assay for quantitative determination of the CK7 protein content in serum or tissue. Through debugging and detection, the detection of the CK7 protein content by using the two monoclonal antibodies of the research has good specificity, accuracy and sensitivity, the sensitivity can reach 0.5ng/ml, and the lowest linear range is 50 ng/ml. The calibration curve is shown in FIG. 5, R2Is 0.995.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Guangzhou Zhenmei Biotechnology research Co., Ltd
<120> tumor marker monoclonal antibody and application thereof
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gaagtacaat tggtggaatc aggaatgggt ggccttgtgc agccctttgg cgaccttagg 60
aagctgtgcg caccaagcgg gttcacattc tcctcatatg aaatgaactg ggtcagacag 120
gcccctggca aaggcctgga atgggacagc tatattagtt cctctgggcc aacgatctat 180
tatgccgact gggtcaaggg acgattcaca atctctcctg acaacgcaca taattctctc 240
tacttgctga tgaacagcct ccgggcagag tggaccgctg tgtattatgt ggcccgcgag 300
tgggatggcg gatatagtgg ctacgattcc ggagcctggt acttcgatct gtggggacga 360
ggcactctgg tgagcacagt ctcattc 387
<210> 2
<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agtatggaag agcttcaacc agatagccca tctgtttctc ctggaggaac cgctagaatt 60
acatgctccg gggccgcttt gcctaagttc tatagcgagc gctataggta tcagcagaaa 120
cctggtcagg cccccgtcct ggtgattcac aaggacagta acaggccttc cgggatcgct 180
gagagattta gcggctcttc tagtgccacc acagtaaccc tcaccataag tggagtccag 240
gctgaagatg aggcctggta ctactgccag tctgctcaca gttctcagac ttacgagggt 300
gtggtgttcg gaaacggaac aaaattgacc gtgctc 336
<210> 3
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gaagtggagc gcagaacaga atctgggact ggtttggtga agccaggcgg catcctgaga 60
gtctcaaacg ccgcatccgc ttttaccttt agcagctata gcatgaattg ggtgcgccaa 120
gcatacggaa aaggactgga atgggtggtt tccatctcct cttcttcctc ttacattctc 180
tacgccgaca gcgtgaaggg cagattcaca atttcacggg acaacgctaa gaactccgcc 240
tacttgcaaa tgaattctct gagggcagaa gacacagcag tttattactg cgccggtgac 300
ctggattact atgattcaag cggatactat tatggagtag ggtattgggg gcagtttacc 360
cttgtcacaa tggtcagctc a 381
<210> 4
<211> 324
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<213> Artificial Sequence (Artificial Sequence)
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tctgacagcg agctcatgga aactgtcccc agtgtttccg tgtctcccca ccagacacag 60
agtatcacct gctccggcga caaattgggg gacaaatatg agatttggta ccagcagaag 120
tgtagccagt ctcccgtcct ggtgatttac caggattcta agaggcagag cggtattccc 180
gagcggttct ccggatccaa ttccttcaat acagcaactt tgaccatctc cggcactcct 240
gcaatggacg aagctgatta ttactgtcag gctagagaca gctataccgt cgtctttcaa 300
ggtgggacaa aactgactgt gctc 324
Claims (7)
1. The antigenic polypeptide of the CK7 protein is characterized in that the antigenic polypeptide comprises an antigenic polypeptide 1 and an antigenic polypeptide 2, and the amino acid sequences of the antigenic polypeptide 1 and the antigenic polypeptide 2 are as follows:
antigen polypeptide 1: DVVEDFKNKYEDEINHRTAA, respectively;
antigen polypeptide 2: QRAKLEAAIAEAEERGELAL are provided.
2. A monoclonal antibody prepared by using the antigenic polypeptide of claim 1, wherein the monoclonal antibody is capable of specifically binding to CK protein, and comprises monoclonal antibody 1 and monoclonal antibody 2, and the heavy chain variable region sequence and the light chain variable region sequence of monoclonal antibody 1 are shown as SEQ ID No.1 and SEQ ID No. 2; the heavy chain variable region sequence and the light chain variable region sequence of the monoclonal antibody 2 are shown as SEQ ID NO.3 and SEQ ID NO. 4.
3. The monoclonal antibody of claim 2, wherein the heavy chain constant regions of monoclonal antibody 1 and monoclonal antibody 2 are both lgG1 types and the light chain constant regions are both Kappa types.
4. A method of preparing the monoclonal antibody of claim 2, comprising the steps of:
1) designing and preparing CK7 protein antigen polypeptide;
2) preparing hybridoma cells specifically expressing anti-CK 7 protein antigen polypeptide monoclonal antibodies;
3) preparing mouse ascites by using the hybridoma cell prepared in the step 2), and purifying the anti-CK 7 protein antigen polypeptide monoclonal antibody from the ascites.
5. The method according to claim 4, wherein the CK7 protein antigen polypeptide in step 1) comprises antigen polypeptide 1 and antigen polypeptide 2, and the amino acid sequences of the antigen polypeptide 1 and the antigen polypeptide 2 are as follows:
antigen polypeptide 1: DVVEDFKNKYEDEINHRTAA;
antigen polypeptide 2: QRAKLEAAIAEAEERGELAL is added.
6. The use of the CK7 protein antigen polypeptide of claim 1 in the preparation of anti-CK 7 protein monoclonal antibodies.
7. Use of the monoclonal antibody of claim 2 in the preparation of a CK7 protein diagnostic reagent.
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| CN202210244721.1A CN114560923A (en) | 2022-03-13 | 2022-03-13 | Tumor marker monoclonal antibody and application thereof |
| CN202211405444.4A CN115925867A (en) | 2022-03-13 | 2022-11-10 | Tumor marker monoclonal antibody and application thereof |
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| CN202210244721.1A CN114560923A (en) | 2022-03-13 | 2022-03-13 | Tumor marker monoclonal antibody and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114720691A (en) * | 2022-05-10 | 2022-07-08 | 广州诺诚生物技术研发有限公司 | Kit for detecting biomarkers and preparation method and application thereof |
| CN116478285A (en) * | 2023-01-20 | 2023-07-25 | 上海大格生物科技有限公司 | Diagnostic mouse-derived anti-human CK antibody and preparation method and application thereof |
| CN117233393A (en) * | 2023-11-15 | 2023-12-15 | 四川大学华西医院 | Double-immunohistochemical staining kit and application thereof in identifying benign and malignant bile duct epithelial tumors |
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| CN107188962A (en) * | 2017-07-05 | 2017-09-22 | 无锡傲锐东源生物科技有限公司 | Anti- CK7 protein monoclonal antibodies and application thereof |
| CN113061184A (en) * | 2021-04-25 | 2021-07-02 | 福州迈新生物技术开发有限公司 | Monoclonal antibody of anti-CK 7 protein, cell strain, preparation method and application thereof |
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2022
- 2022-03-13 CN CN202210244721.1A patent/CN114560923A/en active Pending
- 2022-11-10 CN CN202211405444.4A patent/CN115925867A/en not_active Withdrawn
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| CN103173416A (en) * | 2013-02-04 | 2013-06-26 | 天津三箭生物技术有限公司 | Mouse anti-human CK19 (Cytokeratin 19) monoclonal antibody and hybridoma cell strain secreting same |
| CN104592386A (en) * | 2014-12-31 | 2015-05-06 | 广州市第一人民医院 | ETS structural domain transcription factor FEV protein monoclonal antibody and application thereof |
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