A recombinant monoclonal antibody against Ki-67, its preparation method and application

Abstract

This invention belongs to the field of biotechnology, specifically relating to a recombinant monoclonal antibody against Ki-67, its preparation method, and its applications. The recombinant monoclonal antibody against Ki-67 includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.3; and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.4. This invention utilizes hybridoma technology to prepare a mouse anti-human Ki-67 recombinant monoclonal antibody. This recombinant antibody has a well-defined structure, high titer, and better stability, effectively solving the problems existing in traditional antibody preparation. It can be applied in detection and screening fields such as immunohistochemistry, indirect ELISA, antibody chip preparation, and immunofluorescence, facilitating accurate assessment and detection results.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant monoclonal antibody against Ki-67, its preparation method, and its application. Background Technology

[0002] Antibodies are protective proteins produced by the body in response to antigen stimulation; they are a class of immunoglobulins that specifically bind to antigens. Ki-67 (Actin, aortic smooth muscle) is a nucleoprotein associated with cell proliferation, involved in ribosomal RNA transcription, and plays a crucial role in maintaining cell proliferation. Its expression varies at different stages of the cell cycle, with low expression levels in G1 and early S phases, peaking during mitosis, rapidly declining in late and telophase, and disappearing in G0 phase. It has become a potent indicator for assessing tumor cell proliferation. The Ki-67 index, representing the percentage of Ki-67-stained cells in a tissue, indicates the growth fraction. For many tumors, the cell proliferation rate assessed by Ki-67 immunoreactivity is correlated with tumor grade and clinical course. In recent years, Ki-67 has gradually become a new research hotspot in breast cancer research. Studies show that Ki-67 can be detected by immunohistochemistry, and its expression level can assess the degree of tumor proliferation and thus reflect the invasiveness of breast cancer, especially for early and mid-stage breast cancer patients with negative lymph nodes. It can also be used to assess the prognosis, follow-up plans, and treatment improvement of other malignant tumors. For example, in colorectal cancer, Ki-67 immunohistochemistry can predict the in situ proliferation of tumor cells; Ki-67 > 40% is considered high expression. Colorectal cancer survival rate is negatively correlated with the expression of both Ki-67 and p53 (p53 is a glycoprotein and also a tumor marker), and overexpression of Ki-67 and p53 leads to poor prognosis. In bladder cancer, the Ki-67 index is related to the stage, grade, and size of bladder cancer. The cutoff value for Ki-67 positivity rate is Ki-67 ≥ 20%. Combined detection of Ki-67 and TP53 (TP53 gene, also known as tumor protein p53 gene, is an important tumor suppressor gene) has an important predictive role in the recurrence of non-muscle-invasive bladder cancer.

[0003] Traditional antibody preparation typically involves immunizing animals to obtain monoclonal and polyclonal antibodies. Monoclonal antibodies are produced by immunizing host animals with a target protein, then extracting B cells that recognize and respond to that antigen, fusing them with myeloma cells to form hybridoma cells, thus creating antibodies. Polyclonal antibodies are produced by collecting blood from animals immunized with a target antigen. However, currently prepared antibodies have several problems, such as low antibody titers; the purchased antibody for detecting protein X preferentially binds to protein Y, or even not binds to X at all; poor antibody stability; large batch-to-batch variability; and poor reproducibility, resulting in unrepeatable experimental results when using different batches of antibodies to repeat previous experiments.

[0004] The aforementioned problems have led to erroneous scientific discoveries and wasted research efforts, resulting in significant losses in fields such as cancer, metabolism, aging, immunology, cell signal transduction, and any complex biomolecular research. Therefore, developing a method for preparing anti-Ki-67 antibodies with a well-defined structure, high potency, and high stability is of great importance. Summary of the Invention

[0005] To address the above-mentioned deficiencies or improvement needs of existing technologies, this invention provides a recombinant monoclonal antibody against Ki-67, its preparation method, and its application. The mouse anti-human Ki-67 recombinant monoclonal antibody is prepared by using hybridoma technology to recombinant monoclonal antibodies. This recombinant antibody has a well-defined structure, high potency, and better stability.

[0006] The first aspect of the present invention is to provide a recombinant monoclonal antibody against Ki-67.

[0007] A second aspect of the present invention is to provide a nucleic acid.

[0008] A third aspect of the present invention is to provide a recombinant expression vector.

[0009] The fourth aspect of this invention is to provide a recombinant expression cell.

[0010] The fifth aspect of this invention aims to provide a method for preparing a recombinant monoclonal antibody against Ki-67.

[0011] The sixth aspect of this invention aims to provide the use of the nucleic acid described in the second aspect, or the recombinant expression vector described in the third aspect, or the recombinant expression cell described in the fourth aspect in the preparation of a recombinant monoclonal antibody against Ki-67.

[0012] The seventh aspect of this invention aims to provide the application of the anti-Ki-67 recombinant monoclonal antibody described in the first aspect, or the nucleic acid described in the second aspect, or the recombinant expression vector described in the third aspect, or the recombinant expression cell described in the fourth aspect, or the preparation method described in the fifth aspect, in detecting Ki-67 protein molecules, or in preparing equipment for detecting Ki-67 protein molecules.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, the present invention provides a recombinant monoclonal antibody against Ki-67, the recombinant monoclonal antibody against Ki-67 comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO.3; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO.4.

[0015] In a second aspect, the present invention provides a nucleic acid that encodes the heavy chain variable region and the light chain variable region of the aforementioned anti-Ki-67 recombinant monoclonal antibody.

[0016] Furthermore, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.1-2.

[0017] Furthermore, the nucleotide sequence shown in SEQ ID NO.1 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.1 in the anti-Ki-67 recombinant monoclonal antibody;

[0018] Furthermore, the nucleotide sequence shown in SEQ ID NO.2 is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO.2 in the anti-Ki-67 recombinant monoclonal antibody.

[0019] A third aspect of the present invention provides a recombinant expression vector containing the aforementioned nucleic acid.

[0020] Furthermore, the recombinant expression vector includes a pcDNA3.4 plasmid containing the aforementioned nucleic acid.

[0021] A fourth aspect of the present invention provides a recombinant expression cell containing the above-described nucleic acid or the above-described recombinant expression vector.

[0022] In a fifth aspect, the present invention provides a method for preparing a recombinant monoclonal antibody against Ki-67, wherein the method comprises: transfecting cells with the above-mentioned recombinant expression vector to obtain recombinant expression cells, culturing and transfecting the recombinant expression cells, collecting the supernatant and purifying it to obtain a recombinant monoclonal antibody against Ki-67.

[0023] A sixth aspect of the present invention provides the use of the above-described nucleic acid, or the above-described recombinant expression vector, or the above-described recombinant expression cell in the preparation of a recombinant monoclonal antibody against Ki-67.

[0024] A seventh aspect of the present invention provides the application of the above-described anti-Ki-67 recombinant monoclonal antibody, or the above-described nucleic acid, or the above-described recombinant expression vector, or the above-described recombinant expression cell, or the above-described preparation method in detecting Ki-67 protein molecules, or in preparing an apparatus for detecting Ki-67 protein molecules.

[0025] Furthermore, the detection of Ki-67 protein molecules includes immunohistochemistry, ELISA, and immunofluorescence detection methods.

[0026] Furthermore, the immunohistochemical assay includes: dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB staining, counterstaining, dehydration, mounting, and microscopic examination.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention provides a recombinant monoclonal antibody against Ki-67, its preparation method, and its applications. By utilizing hybridoma technology to recombinant monoclonal antibodies, a recombinant monoclonal antibody encoding the variable region gene of Ki-67 is prepared. The resulting antibody has a well-defined structure, high titer, and good stability. The antibody exhibits high specificity and sensitivity in binding to the Ki-67 protein molecule, specifically recognizing and detecting Ki-67 protein expression in tumor tissues. It shows high positive expression when detecting Ki-67 protein. Therefore, this antibody can be applied in immunohistochemistry, indirect ELISA, antibody chip preparation, immunofluorescence, and other detection and screening fields, facilitating accurate assessment and detection results.

[0029] The present invention provides a recombinant monoclonal antibody against Ki-67, which has the characteristics of good specificity and strong positive signal, and can provide a reference for clinical prognosis assessment. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a flowchart of the preparation and expression process of the anti-Ki-67 recombinant monoclonal antibody of the present invention;

[0032] Figure 2 This is a gel electrophoresis image of total RNA from hybridoma cells in this invention;

[0033] Figure 3 This is a diagram showing the extraction of the variable region coding gene of the anti-Ki-67 recombinant monoclonal antibody of this invention.

[0034] Figure 4 This is a diagram illustrating the construction and verification of the mouse anti-human Ki-67 recombinant antibody gene in this invention.

[0035] Figure 5 This is a diagram of the mouse heavy chain constant region vector pFUSEss-CHIg-mG1 of this invention;

[0036] Figure 6 This is a diagram of the mouse light chain constant region vector pFUSE2ss-CLIg-mk of the present invention;

[0037] Figure 7 This is a diagram showing the preparation results of the Ki-67 protein in this invention;

[0038] Figure 8 This is a graph showing the SDS-PAGE detection results of the mouse anti-human Ki-67 recombinant antibody of this invention;

[0039] Figure 9 This is a sequencing result of the mouse anti-human Ki-67 recombinant antibody sequence P0017-VH1 / VK1 of the present invention;

[0040] Figure 10 The optimal mouse anti-human Ki-67 recombinant antibody sequence P0017-VH1 / VK1 of this invention was detected by pilot-scale SDS-PAGE using the ExpiCHO-S™ Expression System.

[0041] Figure 11 This is a graph showing the titer detection results of the Ki-67 recombinant antibody of this invention;

[0042] Figure 12 This is the affinity constant curve of the Ki-67 recombinant antibody of this invention;

[0043] Figure 13 This is a diagram showing the immunohistochemical verification results of a mouse anti-human Ki-67 recombinant antibody tissue sample from the present invention. Detailed Implementation

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the raw materials used in the following examples are all commercially available products and can be purchased from the market.

[0045] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0046] The raw materials involved in the various embodiments of the present invention are either commercially available products or can be prepared according to existing methods.

[0047] The process flow diagram for the preparation and expression of the anti-Ki-67 recombinant monoclonal antibody of this invention is shown below. Figure 1 As shown.

[0048] The following specific examples will provide further details.

[0049] Example 1: Gene fishing for the variable region of mouse monoclonal antibody

[0050] 1. Results of RNA extraction and reverse transcription from hybridoma cells

[0051] Hybridoma cells were obtained by fusing mouse spleen cells with SP2 / 0 (mouse myeloma cells). Total RNA was extracted from the hybridoma cells using the Trizol method. Clear 28S and 18S bands were observed by agarose gel electrophoresis (e.g., ...). Figure 2 As shown in the figure, the RNA integrity is good. The RNA concentration and purity were measured, and the result was D(260 nm) / D(280 nm) = 1.98, which meets the requirements of this experiment.

[0052] Using total RNA as a template, cDNA was synthesized by reverse transcription using a reverse transcription kit (Tiangen, K118-02). The amplification system and procedure are shown in Tables 1 and 2.

[0053] Table 1 RNA reverse transcription system

[0054]

[0055] Table 2 Program Settings

[0056]

[0057] 2. PCR amplification of the variable region of mouse monoclonal antibody

[0058] Using the cDNA prepared in step 1 as a template, the complete VH gene sequence was amplified by mixing one mouse VH upstream degenerate primer and one VH downstream degenerate primer; the complete VK gene sequence was amplified by mixing two mouse VK upstream degenerate primers and two VK downstream degenerate primers in a certain ratio. The amplification system and procedure are shown in Tables 3 and 4. The agarose gel electrophoresis results of the amplification products are shown below. Figure 3As shown in Figure A (Figure A shows PCR amplification of the heavy / light chain variable region genes: 1: light chain variable region amplification, 2: heavy chain variable region amplification), the VH gene fragment is approximately 350-400 bp in length, and the VK gene fragment is approximately 350 bp in length, consistent with the target fragment length.

[0059] Table 3 PCR amplification system

[0060]

[0061] Table 4 PCR Amplification Program

[0062]

[0063] Table 5 Primer Sequences

[0064]

[0065] 3. Purify the V region gene and ligate it with the T-vector.

[0066] The PCR product from step 2 was purified using a gel extraction kit (Tiangen, DP209-03). The VH and VK genes were ligated into the pGEM-T vector, respectively, and transformed into *E. coli*. Colony PCR was performed using universal primers CMV-F. The target fragment size was approximately 500 bp. Six T clones were selected from the heavy chain, and the colony was identified as correct (Cell P). Six T clones were also selected from the light chain, and the variable region gene clones were identified as correct (Cell P). Results are as follows: Figure 3 B and Figure 3 As shown in Figure C, Figure B shows the identification of T-clone colonies in the heavy chain variable region, with lanes 1-6 representing 1-6 T-clones in the heavy chain variable region. Figure C shows the identification of T-clone colonies in the light chain variable region, with lanes 1-6 representing 1-6 T-clones in the light chain variable region. Six bright banded heavy chain positive clones and four bright banded light chain positive clones (1, 3, 5, and 6) were sent to Kexin Technology Co., Ltd. for sequencing.

[0067] Table 6 Connection Reaction System

[0068]

[0069] 4. Amplification of the full-length recombinant antibody gene

[0070] Using the IMGT / QUEST online analysis software, one valid light chain and one valid heavy chain were screened from the six submitted heavy chain T clones and named P0017-VK1 and P0017-VH1, respectively. The P0017-VK1 light chain gene was analyzed for validity and VDJ family characteristics. The full-length variable region of this functional light chain is 336 bases. The functional light chain belongs to Musmus IGKV1-110. The 01F family showed a 74.49% matching rate in region V and 78.38% in region J. Analysis of the P0017-VH1 heavy chain gene for effectiveness and the VDJ family revealed that the full-length variable region of this functional heavy chain is 336 bases. All functional heavy chains belong to the Musmus IGHV9-2-1 family. For the 01F family, the matching rate for region V is 74.31%, and for region J it is 65.96%. The specific domain divisions of the light chain variable region P0017-VK1 and the heavy chain variable region P0017-VH1 are shown in Tables 7 and 8, respectively. The coding genes for the CDR1, CDR2, and CDR3 domains of the light chain variable region P0017-VK1 and the heavy chain variable region P0017-VH1 are shown in Tables 9 and 10, respectively.

[0071] Table 7. Division of Light Chain Structural Domains in P0017-VK1

[0072]

[0073] Table 8 Division of Heavy Chain Domain in P0017-VH1

[0074]

[0075] Table 9. Light chain variable region structures and their coding genes

[0076]

[0077] Table 10 Heavy chain variable region structures and their coding genes

[0078]

[0079] Example 2: Construction of a full-length recombinant antibody vector against human Ki-67 in mice

[0080] To construct the full-length recombinant antibody plasmid against human Ki-67 in mice, one light chain sequence (P0017-VK1) and one heavy chain sequence (P0017-VH1) were selected and constructed into the pFUSE2ss-CLIg-mk and pFUSEss-CHIg-mG1 vectors, respectively. PCR amplification primers were designed based on the upstream and downstream restriction enzyme sites of the vectors to amplify the full-length recombinant antibody gene. The results are as follows: Figure 4 As shown in Figure A (Figure A shows PCR amplification of the heavy / light chain variable region gene: 1-6: heavy chain variable region amplification, 7-12: light chain variable region amplification), it can be seen from the figure that the size of the Ki-67 heavy chain variable region gene fragment is about 400 bp, and the size of the light chain variable region gene fragment is about 350 bp. The bands are clear, and their molecular size is consistent with the expected theoretical value.

[0081] Subsequently, to determine whether the heavy and light chain variable region genes of the mouse anti-human Ki-67 hybridoma monoclonal antibody were successfully constructed into the pFUSEss-CHIg-mG1 and pFUSE2ss-CLIg-mk vectors, respectively, colony identification was performed on 12 monoclonal clones of the heavy chain variable region and 12 monoclonal clones of the light chain variable region using CMV-F universal primers and heavy chain-specific primers downstream of P0017-VH1-R and light chain-specific primers downstream of P0017-VK1-R. The results are as follows: Figure 4 As shown in Figure B (Figure B shows colony identification of heavy chain variable region gene clones 1-12, where bacteria 3, 4, 5, 8, 10, and 11 are correct (P), and bacteria 13-24 show colony identification of light chain variable region gene clones, where bacteria 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, and 24 are correct (P)), the light chain variable region gene is approximately 500 bp, and the heavy chain variable region gene is approximately 500 bp, consistent with the length of the target fragment. The specific method is as follows:

[0082] 1. PCR amplification primers were designed based on the upstream and downstream restriction enzyme sites of the vector, as shown in Table 11. Then, the heavy and light chain variable region genes that were confirmed to be correct and functional were used as templates by sequencing.

[0083] Table 11 PCR amplification primer sequences

[0084]

[0085] 2. Using mouse heavy chain constant region vector pFUSEss-CHIg-mG1 (e.g., Figure 5 (as shown) and mouse light chain constant region vector pFUSE2ss-CLIg-mk (as shown) Figure 6 Using the above as the backbone, the amplification products of the mouse antibody heavy chain and light chain constant regions were recovered from the gel and then ligated into the mouse heavy chain constant region vector pFUSEss-CHIg-mG1 and the mouse light chain constant region vector pFUSE2ss-CLIg-mk, respectively.

[0086] (1) Light chain constant region modification: The vector pFUSE2ss-CLIg-mk was digested with EcoRI and XhoI to obtain two bands of 3524bp and 336bp in size; the rat light chain Igk constant region gene was amplified, digested with EcoRI and XhoI and ligated into the 3524bp target band, and sequenced for identification and confirmation. The nucleotide sequence encoding the light chain variable region (P0017-VK1) is as follows: GATGTCGTGATGACACAGACCCCTCTGTCCCTGCCTGTGAGCCTGGGCGATCAGGCCAGCATTAGCTGTAGAAGCAGCCAGAGCCTGGTGCACAGCAATGGGAACACCTACCTGCACTGGTATCTGCAGAAAGCCGGGCAGAGCCCCAAGCTACT GATCTACAAGGTGAGCACAGATTCAGCGGCGTGCCCGACCGGTTCAGCGGCAGCGGTCCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAAGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAGAATACTCATGTGCCCCTGACCTTCGGCGCTGGCACCAAGCTTGGAGTTAAAG(SEQ ID NO.2).

[0087] According to the codon encoding rules, the amino acid sequence of the light chain variable region (P0017-VK1) is: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKAGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPLTFGAGTKLELK (SEQ ID NO.4).

[0088] (2) Modification of heavy chain constant region: The vector pFUSEss-CHIg-mG1 was digested with NheI and EcoRI; the mouse heavy chain IgG1 constant region gene was amplified, and the target band was inserted after double digestion with NheI and EcoRI and confirmed by sequencing.

[0089] The nucleotide sequence encoding the first heavy chain variable region (P0017-VH1) is shown below: CAGGTGCAGCTCGTGCAGAGCGGCCCTGAGCTGAAGAAGCCCGGCGAGACCGTGAAGATCTCCTGCAAGGCCAGCGGCTACAGCTTCACCGACCACAGCATGCACTGGGTGAAACAGGCACCCGGAACCGGCCTGAAGTGGATGGGCTGGATCAACACCGAGACCGGCGAGGCCACCTACGCCGACGACTTTAAGGGAAGATTCGCGTTTAGCCTGGAGACAAGCGCCTCCACAGCCTATCTGCAGATTAATAATCTGAAAAATGAGGATACAGCCACTTATTTTGTGTACCTGGGCGATTACTGGGGACAGGGCACCACACTGACCGTGTCCAGC (SEQ ID NO.1);

[0090] According to the codon encoding rules, the amino acid sequence of the first heavy chain variable region (T508-A1T-Ki-67-VH1) is as follows:

[0091] QVQLVQSGPELKKPGETVKISCKASGYSFTDHSMHWVKQAPGTGLKWMGWINTETGEATYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFVYLGDYWGQGTTLTVSS(SEQ ID NO.3);

[0092] Example 3: Expression screening of recombinant monoclonal Ki-67

[0093] 1. Small-scale eukaryotic expression of Expi CHO-S cells

[0094] The four recombinant clones were paired as P0017-VK1 / VH1(R1), and eukaryotic transfection, antibody expression and purification were performed using the ExpiCHO-S™ Expression System in 2 mL batches.

[0095] The specific transfection method based on the ExpiCHO-S™ Expression System is as follows:

[0096] Select a density of 3×10 6 ~1×10 7Seed cells with a viable cell count / mL and a cell viability greater than 95% were used for transfection; the reagent required for transfection was calculated based on the volume of the cell suspension, so that the final cell density in each 2 mL transfection system was 3 × 10⁶ cells / mL. 6 Cells were resuspended at 10% viable cells / mL in Gibco expression medium (approximately 10% of the final volume), and the remaining 90% was brought to the pre-calculated volume with Mirus expression medium. The cells were then incubated at 37°C with appropriate humidity (8% CO2) and a shaking incubator at 125 rpm for 30 min. The transfection reagent was PEI, and the diluent was a NaCl solution containing 5% HEPES. The final cell density was 2 × 10⁶ cells / mL. 6 1 live cells / mL, total volume 2 mL, seeded into a 6-well plate, with one positive control; dilute 4 µg plasmid DNA with 100 µL NaCl, and dilute 10 µg PEI with 100 µL NaCl. Add the diluted plasmid dropwise to each well using a pipette, add one positive control, return to the shaker, and incubate for 5 min. Then add the diluted PEI dropwise to each well, gently shake the 6-well plate, and return to the shaker for incubation at 37°C with appropriate humidity, 8% CO2, and a rotation speed of 125 rpm. 24 hours after transfection, add 50 µL sodium butyrate and 100 µL EN3 to each well. After approximately 72 h, start sampling from the culture medium to detect recombinant protein expression.

[0097] Purification was performed using pure beans, and the specific steps are as follows:

[0098] Take 100 µL of pure beads (Protein A, a membrane protein) into a 1.5 mL EP tube, wash twice with 1 mL PBS, and add 400 µL PBS buffer to prepare 20% beads. Take eukaryotic transfected cells, centrifuge at 1000 rpm for 5 min, take 40 µL of supernatant sample, take an appropriate amount of precipitate sample, and dilute with 40 µL PBS buffer. Collect the supernatant, add 50 µL of beads, and incubate at room temperature for 30 min. Collect the incubated beads, and gradually transfer the supernatant after incubation to a new 1.5 mL EP tube, centrifuge, take 40 µL of supernatant as flow-through sample, discard the rest of the flow-through, and repeat this step to collect all beads. Add 10 µL of 5×loading buffer to the supernatant, precipitate, and flow-through, and add 20 µL of 5×loading buffer to the beads. Boil all samples at 100℃ for 10 min and perform SDS-PAGE gel electrophoresis.

[0099] The results are as follows Figure 8As shown, after eukaryotic transfection and antibody expression purification using the ExpiCHO-S™ Expression System (2 mL), the reduced SDS-PAGE electrophoresis showed clear bands at 25 kDa and 55 kDa without any extraneous bands, consistent with the expected target bands.

[0100] 2. Ki-67 protein preparation

[0101] The primer sequences for Ki-67 were designed using the UNIPROT (https: / / www.uniprot.org / ) and NCBI (https: / / www.ncbi.nlm.nih.gov / ) websites as follows: Ki-67 upstream primer: GGATCTTCCAGAGATATGTGGCCCACGAGACGC; Ki-67 downstream primer: CTGCCGTTCGACGATTGTAGGAATACTTCCTCTGGTTTTGG; primers were synthesized by Qingke Biotechnology. The target fragment of Ki-67 was amplified by polymerase chain reaction (PCR). After amplification, 1 µL of the recombinant product of Ki-67 was verified by agarose gel electrophoresis, and the target fragment of Ki-67 was recovered using an agarose gel recovery kit. The expression vector pET21b was digested with restriction endonucleases BamHI and XhoI. Subsequently, the target fragment of Ki-67 was recombined with the linearized vector and transformed into competent BL21 cells. White single colonies were picked for sequence identification. Figure 7 Figure A shows the Ki-67 colony PCR agarose gel electrophoresis image.

[0102] The correctly identified clone was cultured in Luria-Bertani LB medium containing ampicillin, followed by protein expression induction at 37°C with 0.5 mmol / L IPTG (isopropyl-β-D-thiogalactopyranoside). The bacterial cells were collected, sonicated, and the supernatant was collected. The supernatant was added to nickel affinity packing material at a controlled flow rate, followed by elution with imidazole eluent at concentrations of 20 mmol / L, 50 mmol / L, 100 mmol / L, 250 mmol / L, and 500 mmol / L. 20 µL of the flow-through and different concentrations of imidazole eluent were subjected to denaturing and reducing polyacrylamide gel electrophoresis. The protein eluted with 50 mmol / L imidazole eluent was concentrated and subjected to non-denaturing, non-reducing polyacrylamide gel electrophoresis. The electrophoresis results are shown below. Figure 7 Figure B shows the Ki-67 polyacrylamide gel electrophoresis diagram.

[0103] 3. ELISA detection of the titer of mouse anti-human Ki-67 recombinant antibody

[0104] To further screen for the optimal mouse anti-human Ki-67 recombinant antibody sequence, a 2 mL eukaryotic transfection and antibody expression purification were performed using the ExpiCHO-S™ Expression System, followed by ELISA detection of the supernatant titer.

[0105] The specific methods for determining potency are as follows:

[0106] 1) Coating: Prepare carbonate buffer solution, dilute Ki-67 protein to 2 µg / mL, add 100 µL / well of diluted Ki-67 protein to a 96-well microplate, and then react overnight at 4ºC;

[0107] 2) Washing the plate: Pour out the liquid from the 96-well microplate, wash the plate 3 times with PBST buffer, and then pat the liquid in the 96-well microplate dry on filter paper;

[0108] 3) Blocking: Add 200 µL of 2% BSA (bovine serum albumin) to each well and incubate at 37°C for 2 hours;

[0109] 4) Wash the plate: Repeat step 2).

[0110] 5) Add primary antibody: Set antibody concentrations of 10 ng / mL, 3.33 ng / mL, and 1.11 ng / mL. Dilute the antibody according to these three concentration gradients. Add 100 µL of diluted antibody (the anti-Ki-67 recombinant monoclonal antibody prepared in this invention) to each well and incubate at 37°C for 1 hour.

[0111] 6) Wash the board: Repeat step 2).

[0112] 7) Add enzyme-labeled secondary antibody: Add 100 µL of 1:4000 enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) to each well and incubate at 37℃ for 40 min;

[0113] 8) Wash the plate: Repeat step 2).

[0114] 9) Color development: Add 100 µL of color development solution to each well, place in an incubator, incubate at 37℃ for 10 min, and add 50 µL of 2mol / L sulfuric acid solution to terminate the reaction;

[0115] 10) OD value determination: Use an ELISA reader to determine the absorbance (OD 450) value of each well.

[0116] The results showed that the P0017-VK1 / VH1(R1) ELISA results were as follows: Figure 11 As shown, the result is positive with a strong signal, which can effectively identify the coated Ki-67 protein.

[0117] The P0017-VK1 / VH1(R1) bacterial culture was sent to a sequencing company for sequencing analysis. The sequencing results are as follows: Figure 9 As shown, the results indicate that the sequencing peaks are distinct, clear, and symmetrical, with good resolution and signal-to-noise ratio, and no abnormal or extraneous peaks, indicating that the sequencing results are reliable and can be used for subsequent experiments.

[0118] To further obtain milligram-level Ki-67 recombinant antibodies for sample validation and characterization, this study used the ExpiCHO-S™ Expression System to perform 60 mL eukaryotic pilot-scale expression of P0017-VK1 / VH1(R1) and antibody affinity chromatography purification. The purified recombinant antibody was analyzed by reducing SDS-PAGE (e.g., ...). Figure 10 As shown in the figure, there are clear and distinct bands at 25 kDa and 55 kDa without any extraneous bands, which is consistent with the expected target band.

[0119] Example 4: Identification of recombinant monoclonal Ki-67

[0120] This embodiment describes the ELISA affinity detection of the mouse anti-human Ki-67 recombinant monoclonal antibody prepared according to the methods described in Examples 1-3. The detection process is as follows:

[0121] To further characterize the affinity of the Ki-67 recombinant antibody, an indirect ELISA method was used for identification, followed by OD... 450 Using nm as the ordinate and the logarithm of antibody concentration as the abscissa, two curves were fitted using Origin 8.5 software to calculate the OD. max The antibody affinity constant Ka was calculated based on the antibody concentration (mol / L) corresponding to / 2. The results are as follows: Figure 12 As shown, A represents the concentration of the coated recombinant protein (0.2 μg / mL), and B represents the concentration of the coated recombinant protein (0.01 μg / mL).

[0122] According to the affinity constant Ka formula:

[0123] (Equation 1-1)

[0124] In the formula, [Ab]1 and [Ab]2 are the antibody concentrations (mol / L) corresponding to two different coating concentrations at ODmax / 2. [Ab]1 corresponds to a relatively low coating concentration, and [Ab]2 corresponds to a relatively high coating concentration. n is the ratio of the two coating concentrations (n>1).

[0125] The affinity of the Ki-67 recombinant antibody was calculated, and the results showed that the affinity of the Ki-67 recombinant antibody was 6.6 × 10⁻⁶. 10 L / mol, affinity greater than 10 7L / mol indicates a high affinity antibody, suggesting that the Ki-67 recombinant antibody prepared in this invention has high affinity.

[0126] Example 5:

[0127] This embodiment describes the immunohistochemical detection of the anti-Ki-67 recombinant monoclonal antibody prepared according to the methods described in Examples 1-3. The detection process is as follows:

[0128] Ki-67 is located in the cell nucleus, and its proliferation index is closely related to the differentiation, invasion, metastasis, and prognosis of many tumors. Therefore, it is widely used as a marker for various malignant tumors and in tumor research. To further verify the recognition ability of the Ki-67 recombinant antibody on Ki-67 protein expression in pathological tissue samples, this example uses immunohistochemical detection of the anti-Ki-67 recombinant monoclonal antibody. The detection process is as follows:

[0129] (1) Baking: Place the paraffin slices in a 65℃ oven for 2-3 hours to allow the paraffin to melt completely;

[0130] (2) Dewaxing and hydration: ① Dewaxing with xylene, passing through three tanks of xylene for 5 minutes each time; ② Hydration: first passing through two tanks of 100% ethanol for 5 minutes each time, then passing through 95% ethanol for 5 minutes, 70% ethanol for 5 minutes, and finally rinsing with two tanks of pure water for 5 minutes (machine hydration takes about 50 minutes).

[0131] (3) Antigen retrieval: ① Citrate high-pressure antigen retrieval (100x, pH 6.0): Prepare fresh and use immediately. The retrieval solution must completely submerge the slide. First, boil the tap water in the pressure cooker, then put the retrieval box (including the high-temperature staining rack) into it, heat to boiling, first place the slide on the steam to moisten it, then put the slide into the retrieval solution, close the pressure valve, heat until steam is released, start timing for 5 minutes, disconnect the power, take out the retrieval box, place at room temperature for 20 minutes, and take out the slide after natural cooling; ② EDTA high-temperature antigen retrieval (50x, pH 9.0): Prepare fresh and use immediately. First, boil the tap water, then put it into the retrieval box, heat to boiling, then put in the slide, do not close the pressure valve, start timing for 20 minutes, disconnect the power, take out the retrieval box, place at room temperature for 20 minutes, and take out the slide after natural cooling;

[0132] (4) Washing: Rinse with pure water for 5 min × 2, then rinse with PBS buffer for 5 min × 2;

[0133] (5) Blocking: Add 20-30 μL of 10% BSA to a humidified chamber and block at 37°C for 60 min. Discard the blocking solution.

[0134] (6) Primary antibody incubation: Add 20-30 μL of primary antibody (anti-Ki-67 recombinant monoclonal antibody prepared in this invention) diluted in an appropriate ratio, and incubate in a humidified chamber at 37°C for 1 h; or at 4°C overnight; first add PBS buffer to dilute the primary antibody, and then wash with PBS buffer;

[0135] (7) Secondary antibody incubation: Wash with PBS buffer for 10 min × 3, add 20-30 uL of secondary antibody (HRP-rabbit anti-mouse IgG) diluted appropriately, and incubate in a humidified chamber at 37℃ for 1 h; rinse with PBS for 5 min × 2;

[0136] (8) DAPI staining of the nucleus and mounting: Observe under fluorescence, take pictures, and if necessary, store in a dark box at 4°C;

[0137] Some immunohistochemical test results, such as Figure 13 As shown in Figure A (immunohistochemical detection of human tonsils with Ki-67 recombinant antibody) and Figure B (immunohistochemical detection of human liver cancer with Ki-67 recombinant antibody): Ki-67 is located in the cell nucleus, which is consistent with the expected tissue localization, tissue sublocalization, cellular localization and subcellular localization. The signal is strong and is expected to be used for clinical diagnosis.

[0138] In summary, this invention successfully prepared a mouse anti-human Ki-67 recombinant monoclonal antibody with high titer and affinity. The immunohistochemical detection results of the Ki-67 recombinant antibody were similar to those of the commercial Ki-67 antibody, and in some tissue samples, the positive rate of immunohistochemical detection was significantly higher than that of the commercial Ki-67 antibody. This antibody can be used for immunohistochemical detection and can provide preliminary data support for clinical pathological diagnosis.

[0139] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant monoclonal antibody against Ki-67, characterized in that, The anti-Ki-67 recombinant monoclonal antibody includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.3; The light chain variable region includes the amino acid sequence shown in SEQ ID NO.

4.

2. A nucleic acid, characterized in that, The nucleic acid encodes the heavy chain variable region and the light chain variable region of the anti-Ki-67 recombinant monoclonal antibody as described in claim 1.

3. A nucleic acid according to claim 2, characterized in that, The nucleic acid includes the nucleotide sequence shown in SEQ ID NO.1-2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid as described in any one of claims 2-3.

5. A recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector includes the pcDNA3.4 plasmid.

6. A recombinant expression cell, characterized in that, The recombinant expression cells contain the nucleic acid as described in any one of claims 2-3, or the recombinant expression vector as described in claim 4 or 5.

7. A method for preparing a recombinant monoclonal antibody against Ki-67, characterized in that, The preparation method is as follows: recombinant expression cells are obtained by transfecting cells with the recombinant expression vector as described in claim 4 or 5, and the recombinant expression cells are obtained after culture and transfection. The supernatant is collected and purified to obtain the anti-Ki-67 recombinant monoclonal antibody.

8. The use of a nucleic acid as described in any one of claims 2-3, or a recombinant expression vector as described in claim 4 or 5, or a recombinant expression cell as described in claim 6 in the preparation of a recombinant monoclonal antibody against Ki-67.

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