Anti-Ki-67 recombinant monoclonal antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-Ki-67 recombinant monoclonal antibody as well as a preparation method and application thereof. The anti-Ki-67 recombinant monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO.3; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO.4. The mouse anti-human Ki-67 recombinant monoclonal antibody is prepared by using a hybridoma technology recombinant monoclonal antibody, and the recombinant antibody is clear in structure, high in titer and better in stability, can effectively solve the problems existing in traditional antibody preparation, can be applied to the detection and screening fields of immunohistochemistry, indirect ELISA, antibody chip preparation, immunofluorescence and the like, and has good application prospects. And accurate evaluation and detection results can be obtained.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-Ki-67 recombinant monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Antibodies refer to proteins with protective effects produced by the body due to antigen stimulation. They are a class of immunoglobulins that can specifically bind to antigens. Ki-67 (Actin, aortic smooth muscle, Ki-67) is a nuclear protein associated with cell proliferation, associated with ribosomal RNA transcription, and plays a key role in maintaining cell proliferation. It is expressed differently in different stages of the cell cycle, with a low expression level in the G1 phase and early S phase, a peak in the mitotic phase, a rapid decline in the late and terminal phases, and no expression in the G0 phase, becoming a strong indicator for evaluating the proliferation index of tumor cells. The Ki-67 index, that is, the percentage of cells stained with Ki-67 in the tissue, represents the growth score. For many tumors, the cell proliferation rate assessed by Ki-67 immunoreactivity is related to the tumor grade and clinical course. In recent years, Ki-67 has gradually become a new research hotspot in breast cancer research. Studies have shown that Ki-67 can be detected by immunohistochemistry, and its expression value can evaluate the degree of tumor proliferation and thus reflect the invasiveness of breast cancer, especially for patients with early and mid-stage breast cancer with negative lymph nodes. It can also be used to evaluate the prognosis, follow-up plan and improve treatment of other malignant tumors. For example, in colorectal cancer, Ki-67 immunohistochemistry can predict the proliferation function of tumor cells in situ, and Ki-67>40% is considered to be high expression; the survival rate of colorectal cancer is negatively correlated with the expression of Ki-67 and p53 (p53 is a glycoprotein and also a tumor marker), and overexpression of Ki-67 and p53 will lead to poor prognosis. In bladder cancer, the Ki-67 index is related to the stage, grade and tumor size of bladder cancer. The critical value of Ki-67 positivity rate is Ki-67 ≥ 20%. The combined detection of Ki-67 and TP53 (TP53 gene, also known as tumor protein p53 gene, is an important tumor suppressor gene) plays an important predictive role in the recurrence of non-muscle invasive bladder cancer.

[0003] Traditional antibody preparation generally obtains monoclonal antibodies and polyclonal antibodies by immunizing animals. Monoclonal antibodies are prepared by immunizing host animals with target proteins, extracting B cells that can recognize and respond to the antigen, and fusing them with myeloma cells to form hybridoma cells. Polyclonal antibodies are made by collecting blood from animals that have been immunized with target antigens. However, there are many problems with the antibodies currently prepared, such as low antibody titer, the purchased antibodies used to detect protein X preferentially bind to protein Y, or even do not bind to X at all; the antibody stability is poor, the batch differences are large, and the experimental results cannot be repeated when repeating previous experiments with different batches of antibodies, and the repeatability is poor.

[0004] The above problems have led to erroneous scientific discoveries and wasted scientific research efforts, resulting in huge losses in cancer, metabolism, aging, immunology and cell signal transduction, as well as any field of complex biological molecules. Therefore, it is of great significance to develop a method for preparing an anti-Ki-67 antibody with a clear structure, high potency and high stability. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides an anti-Ki-67 recombinant monoclonal antibody and a preparation method and application thereof. Mouse anti-human Ki-67 recombinant monoclonal antibody is prepared by recombinant monoclonal antibody using hybridoma technology. The recombinant antibody has a clear structure, high titer and better stability.

[0006] The first aspect of the present invention aims to provide an anti-Ki-67 recombinant monoclonal antibody.

[0007] The second aspect of the present invention aims to provide a nucleic acid.

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

[0009] The fourth aspect of the present invention aims to provide a recombinant expression cell.

[0010] The fifth aspect of the present invention is to provide a method for preparing an anti-Ki-67 recombinant monoclonal antibody.

[0011] The sixth aspect of the present invention is to provide a 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 anti-Ki-67 recombinant monoclonal antibodies.

[0012] The object of the seventh aspect of the present invention is to provide an 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 a device for detecting Ki-67 protein molecules.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is: In a first aspect of the present invention, an anti-Ki-67 recombinant monoclonal antibody is provided, wherein the anti-Ki-67 recombinant monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.3; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.4.

[0014] The second aspect of the present invention provides a nucleic acid encoding the heavy chain variable region and the light chain variable region of the above-mentioned anti-Ki-67 recombinant monoclonal antibody.

[0015] Furthermore, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.1-2.

[0016] 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; 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.

[0017] The third aspect of the present invention provides a recombinant expression vector, which contains the above-mentioned nucleic acid.

[0018] Furthermore, the recombinant expression vector includes a pcDNA3.4 plasmid containing the above-mentioned nucleic acid.

[0019] The fourth aspect of the present invention provides a recombinant expression cell, wherein the recombinant expression cell contains the above-mentioned nucleic acid or the above-mentioned recombinant expression vector.

[0020] The fifth aspect of the present invention provides a method for preparing an anti-Ki-67 recombinant monoclonal antibody, the preparation method comprising: using the above-mentioned recombinant expression vector to transfect cells to obtain recombinant expression cells, culturing the recombinant expression cells obtained after transfection, collecting and purifying the supernatant to obtain the anti-Ki-67 recombinant monoclonal antibody.

[0021] The sixth aspect of the present invention provides a use of the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant expression cell in the preparation of anti-Ki-67 recombinant monoclonal antibodies.

[0022] The seventh aspect of the present invention provides an application of the above-mentioned anti-Ki-67 recombinant monoclonal antibody, or the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant expression cell, or the above-mentioned preparation method in detecting Ki-67 protein molecules, or preparing a device for detecting Ki-67 protein molecules.

[0023] Furthermore, the detection of Ki-67 protein molecules includes immunohistochemistry, ELISA, and immunofluorescence detection methods.

[0024] Furthermore, the immunohistochemical detection includes: dewaxing, antigen repair, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB color development, counterstaining, dehydration, sealing and microscopic examination.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an anti-Ki-67 recombinant monoclonal antibody and a preparation method and application thereof. The recombinant monoclonal antibody variable region encoding gene of anti-Ki-67 is prepared by utilizing hybridoma technology to recombinant monoclonal antibodies, and the obtained antibody has a clear structure, high titer and good stability. The antibody binds to the Ki-67 protein molecule with high specificity and high sensitivity, can specifically identify and detect the expression of Ki-67 protein on tumor tissue, and is positively highly expressed when detecting Ki-67 protein. Therefore, the antibody can be applied to detection and screening fields such as immunohistochemistry, indirect ELISA, antibody chip preparation, and immunofluorescence, which is conducive to obtaining accurate evaluation and detection results.

[0026] The present invention provides an anti-Ki-67 recombinant monoclonal antibody, which has the characteristics of good specificity, strong positive signal, etc., and can provide a reference for clinical prognosis evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 It is a process flow chart of the preparation and expression of the anti-Ki-67 recombinant monoclonal antibody of the present invention; Figure 2 is a gel electrophoresis diagram of total RNA of hybridoma cells of the present invention; Figure 3 It is a map of the variable region encoding gene of the anti-Ki-67 recombinant monoclonal antibody of the present invention; Figure 4 This is a verification diagram of the gene construction of the mouse anti-human Ki-67 recombinant antibody of the present invention; Figure 5 This is a diagram of the mouse heavy chain constant region vector pFUSEss-CHIg-mG1 of the present invention; Figure 6 This is a diagram of the mouse light chain constant region vector pFUSE2ss-CLIg-mk of the present invention; Figure 7 This is a diagram showing the preparation results of the Ki-67 protein of the present invention; Figure 8 This is a graph showing the results of SDS-PAGE detection of the mouse anti-human Ki-67 recombinant antibody of the present invention; Fig. 9 It is a sequence diagram of the mouse anti-human Ki-67 recombinant antibody sequence P0017-VH1 / VK1 of the present invention; Fig.10 This is the result of the pilot SDS-PAGE test of the optimal mouse anti-human Ki-67 recombinant antibody sequence P0017-VH1 / VK1 using the ExpiCHO-S™ Expression System; Fig.11 This is a graph showing the titer test results of the Ki-67 recombinant antibody of the present invention; Fig.12 is the affinity constant curve of the Ki-67 recombinant antibody of the present invention; Fig.13 This is a diagram showing the immunohistochemical validation results of mouse anti-human Ki-67 recombinant antibody tissue samples of the present invention. DETAILED DESCRIPTION

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified.

[0030] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form a new or preferred technical solution.

[0031] The raw materials involved in the embodiments of the present invention are either existing commercially available products or can be prepared according to existing methods.

[0032] The process flow chart of the preparation and expression of the anti-Ki-67 recombinant monoclonal antibody of the present invention is as follows: Figure 1 shown.

[0033] The following is further described with reference to specific embodiments.

[0034] Example 1: Mouse monoclonal antibody variable region gene fishing 1. Hybridoma cell RNA extraction and reverse transcription results Mouse spleen cells and SP2 / 0 (mouse myeloma cells) were fused to obtain hybridoma cells. The total RNA of hybridoma cells was extracted by Trizol method. Clear 28S and 18S bands were observed by agarose gel electrophoresis (e.g. Figure 2 The RNA concentration and purity were measured, and the result was D(260 nm) / D(280 nm) = 1.98, which can meet the requirements of this experiment.

[0035] Total RNA was used as a template to synthesize cDNA by reverse transcription using a reverse transcription kit (Tiangen, K118-02). The amplification system and procedure are shown in Tables 1 and 2.

[0036] Table 1 RNA reverse transcription system Table 2 Program settings 2. PCR amplification of mouse monoclonal antibody variable region Using the cDNA prepared in step 1 as a template, one mouse VH upstream degenerate primer and one VH downstream degenerate primer were used to amplify the full set of VH genes; two mouse VK upstream degenerate primers and two VK downstream degenerate primers were mixed in a certain ratio to amplify the full set of VK genes. The amplification system and procedure are shown in Tables 3 and 4. The amplification product agarose gel electrophoresis results are shown in Figure 3 As shown in A (Figure A is the PCR amplification of the heavy chain / light chain variable region genes: 1: light chain variable region amplification, 2: heavy chain variable region amplification), the length of the VH gene fragment is approximately 350-400 bp, and the length of the VK gene fragment is approximately 350 bp, which is consistent with the length of the target fragment.

[0037] Table 3 PCR amplification system Table 4 PCR amplification program Table 5 Primer sequences 3. Purification of V region genes and ligation with T-vector Purify the PCR product of step 2 with a gel recovery kit (Tiangen, DP209-03), connect the VH gene and VK gene to the pGEM-T vector, and transform E. coli. Use universal primer CMV-F for colony PCR. The target fragment size is about 500bp. Select 6 T clones for the heavy chain, and the strain P is correct. Select 6 T clones for the light chain. Variable region gene clone colony identification, strain P is correct. The results are as follows Figure 3 B and Figure 3 C, where Figure B is the identification of T clone colonies in the heavy chain variable region, and lanes 1-6 represent T clones 1-6 in the heavy chain variable region. Figure C is the identification of T clone colonies in the light chain variable region, and lanes 1-6 represent T clones 1-6 in the light chain variable region. The 6 positive clones of the heavy chain with bright bands and the 4 positive clones of the light chain 1, 3, 5, and 6 were sent to Qingke Company for sequencing.

[0038] Table 6 Ligation reaction system 4. Full-length recombinant antibody gene amplification The IMGT / QUEST online analysis software was used to screen out 1 effective light chain and 1 effective heavy chain from the 6 heavy chain T clones and 6 light chain T clones, which were named P0017-VK1 and P0017-VH1, respectively. The light chain P0017-VK1 gene was analyzed for validity and VDJ family. The full length of the functional light chain variable region was 336 bases, and the functional light chain belonged to Musmus IGKV1-110. 01 F family, the V region match rate was 74.49%, and the J region match rate was 78.38%. The heavy chain P0017-VH1 gene was analyzed for validity and VDJ family. The functional heavy chain variable region was 336 bases long, and the functional heavy chains all belonged to Musmus IGHV9-2-1 01 F family, the V region matching rate was 74.31%, and the J region was 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 of 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.

[0039] Table 7 P0017-VK1 light chain domain division Table 8 P0017-VH1 heavy chain domain division Table 9 Light chain variable region domains and encoding genes Table 10 Heavy chain variable region domains and encoding genes Example 2: Construction of mouse anti-human Ki-67 full-length recombinant antibody vector In order to construct mouse anti-human Ki-67 full-length recombinant antibody plasmid, one light chain sequence (P0017-VK1) and one heavy chain sequence (P0017-VH1) obtained by screening were constructed into pFUSE2ss-CLIg-mk and pFUSEss-CHIg-mG1 vectors, respectively. PCR amplification primers were designed based on the upstream and downstream restriction sites of the vectors to amplify the full-length recombinant antibody gene. The results are shown in Figure 2. Figure 4 As shown in Figure A (Figure A is the heavy chain / light chain variable region gene PCR amplification: 1-6: heavy chain variable region amplification, 7-12: light chain variable region amplification), it can be seen from the figure that the Ki-67 heavy chain variable region gene fragment size is about 400 bp, the light chain variable region gene fragment size is about 350 bp, the bands are clear, and the molecular size is consistent with the expected theoretical value.

[0040] Subsequently, in order to identify whether the heavy chain and light chain variable region genes of mouse anti-human Ki-67 hybridoma monoclonal antibodies were successfully constructed into pFUSEss-CHIg-mG1 and pFUSE2ss-CLIg-mk vectors, respectively, 12 monoclonal heavy chain variable region clones and 12 monoclonal light chain variable region clones were identified using CMV-F universal primers and the downstream of the heavy chain specific primer P0017-VH1-R and the downstream of the light chain specific primer P0017-VK1-R. The results are as follows: Figure 4 As shown in Figure B (Figure B shows colony identification of clones of heavy chain variable region gene clones 1-12, of which 3, 4, 5, 8, 10, 11 strains are correct, and 13-24 are colony identification of clones of light chain variable region gene clones, of which 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24 strains are correct), the light chain variable region gene is about 500 bp, and the heavy chain variable region gene is about 500 bp, which is consistent with the target fragment length. The specific method is as follows: 1. Design PCR amplification primers based on the upstream and downstream restriction sites of the vector as shown in Table 11, and then use the heavy chain and light chain variable region genes that are correct and functional as templates confirmed by sequencing.

[0041] Table 11 PCR amplification primer sequences 2. Use mouse heavy chain constant region vector pFUSEss-CHIg-mG1 (such as Figure 5 ) and the mouse light chain constant region vector pFUSE2ss-CLIg-mk (as Figure 6The amplified products of the heavy chain and light chain constant regions of the above mouse antibodies were recovered by gel and connected to the mouse heavy chain constant region vector pFUSEss-CHIg-mG1 and the mouse light chain constant region vector pFUSE2ss-CLIg-mk respectively.

[0042] (1) Light chain constant region modification: The vector pFUSE2ss-CLIg-mk was double-digested with EcoRI and XhoI to obtain two bands of 3524 bp and 336 bp respectively; the rat light chain Igk constant region gene was amplified, double-digested with EcoRI and XhoI, and then connected to the 3524 bp 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).

[0043] According to the codon encoding rules, the amino acid sequence of the light chain variable region (P0017-VK1) is: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKAGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPLTFGAGTKLELK (SEQ ID NO.4).

[0044] (2) Heavy chain constant region modification: The vector pFUSEss-CHIg-mG1 was double-digested with NheI and EcoRI; the mouse heavy chain IgG1 constant region gene was amplified, double-digested with NheI and EcoRI, and then connected to the target band, and sequenced for identification and confirmation.

[0045] The nucleotide sequence encoding the first heavy chain variable region (P0017-VH1) is as follows: CAGGTGCAGCTCGTGCAGAGCGGCCCTGAGCTGAAGAAGCCCGGCGAGACCGTGAAGATCTCCTGCAAGGCCAGCGGCTACAGCTTCACCGACCACAGCATGCACTGGGTGAAACAGGCACCCGGAACCGGCCTGAAGTGGATGGGCTGGATCAACACCGAGACCGGCGAGGCCACCTACGCCGACGACTTTAAGGGAAGATTCGCGTTTAGCCTGGAGACAAGCGCCTCCACAGCCTATCTGCAGATTAATAATCTGAAAAATGAGGATACAGCCACTTATTTTGTGTACCTGGGCGATTACTGGGGACAGGGCACCACACTGACCGTGTCCAGC (SEQ ID NO. 1); According to the codon encoding rules, the amino acid sequence of the first heavy chain variable region (T508-A1T-Ki-67-VH1) is: QVQLVQSGPELKKPGETVKISCKASGYSFTDHSMHWVKQAPGTGLKWMGWINTETGEATYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFVYLGDYWGQGTTLTVSS(SEQ ID NO.3); Example 3: Expression screening of recombinant monoclonal Ki-67 1. Expi CHO-S cell eukaryotic expression test The four recombinant clones were paired as P0017-VK1 / VH1(R1), and the ExpiCHO-S™ Expression System was used for 2 mL eukaryotic small-scale transfection and antibody expression and purification.

[0046] The specific transfection method based on ExpiCHO-S™ Expression System is as follows: Select a density of 3×10 6 ~1×10 7 The seed cells were transfected when the cell viability was greater than 95% and the reagents required for transfection were calculated according to the volume of the cell suspension, so that the final cell density in each 2 mL transfection system was 3×10 6The cells were resuspended in Gibco expression medium (accounting for about 10% of the final volume), and the remaining 90% was filled up to the pre-calculated volume with Mirus expression medium. The cells were cultured in a shaking incubator at 37°C, with appropriate humidity, 8% CO2, and a speed of 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 2×10 6 viable cells / mL, total volume 2 mL, six-well plates were plated, and a positive control was set up; 4 µg plasmid DNA was diluted with 100 µL NaCl, and 10 µg PEI was diluted with 100 µL NaCl. The diluted plasmid was added dropwise to each well with a pipette to set up a positive control, and then returned to the shaker. After incubation for 5 min, the PEI dilution was added dropwise to each well, and the six-well plates were gently shaken and returned to the shaker for culture in a 37°C shaking incubator with appropriate humidity, 8% CO2, and a speed of 125 rpm; 24 hours after transfection, 50 µL sodium butyrate and 100 µL EN3 were added to each well. After about 72 h, samples were taken from the culture medium to detect the expression of the recombinant protein.

[0047] Purification was performed using pure beads, and the specific steps are as follows: Take 100 µL of pure beads (Protein A, a membrane protein) to a 1.5 mL EP tube, wash twice with 1 mL PBS, and add 400 µL PBS buffer to prepare 20% beads; take the 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 it 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, gradually transfer the incubated supernatant to a new 1.5 mL EP tube, centrifuge, take 40 µL of supernatant as the flow-through sample, discard the rest of the flow-through, repeat this step, and collect all beads; add 10 µL 5×loading Buffer to the supernatant, precipitate, and flow-through, and add 20 µL 5×loading Buffer to the beads; boil all samples at 100°C for 10 min, and perform SDS-PAGE gel electrophoresis detection.

[0048] The results are as follows Figure 8 As shown, after using the ExpiCHO-S™ Expression System for 2 mL eukaryotic cell transfection and antibody expression and purification, the reducing SDS-PAGE electrophoresis analysis showed clear bands at 25 kDa and 55 kDa with no other bands, which was consistent with the expected target bands.

[0049] 2. Preparation of Ki-67 protein The primer sequences of Ki-67 were designed through 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; the primers were synthesized by Qingke Bio. The target fragment of Ki-67 was amplified by polymerase chain reaction. After amplification, 1 µL of the recombinant product of Ki-67 was taken for agarose gel electrophoresis verification, and the Ki-67 target fragment was recovered using an agarose gel recovery kit. The expression vector pET21b was digested with restriction endonucleases BamH I and XhoI, and then the target fragment of Ki-67 and the linearized vector were recombined and transformed into competent cells BL21, and white single colonies were picked for sequence identification as shown in Figure 7 As shown in A (Figure A is the agarose gel electrophoresis image of Ki-67 colony PCR).

[0050] Take the bacterial liquid of the correctly identified cloned strain and expand it in LB medium (Luria-Bertani medium) containing ampicillin, and then induce protein expression under the conditions of 0.5 mmol / L IPTG (isopropyl-β-D-thiogalactoside) and 37°C. Collect the bacteria, use an ultrasonic disruptor to disrupt the bacteria and collect the supernatant. Add the supernatant to the nickel affinity filler at a certain flow rate, and then elute the protein with 20 mmol / L, 50 mmol / L, 100 mmol / L, 250 mmol / L and 500 mmol / L imidazole elution buffers in sequence. Take 20 µL of the flow-through and elution buffers of different concentrations of imidazole for denaturing reduced polyacrylamide gel electrophoresis. Concentrate the protein eluted with 50 mmol / L imidazole elution buffer and perform non-denaturing non-reducing polyacrylamide gel electrophoresis. The electrophoresis results are as follows. Figure 7 As shown in B (Figure B is the Ki-67 polyacrylamide gel electrophoresis diagram).

[0051] 3. Mouse anti-human Ki-67 recombinant antibody ELISA test titer In order to further screen the optimal mouse anti-human Ki-67 recombinant antibody sequence, the ExpiCHO-S™ Expression System was used for 2 mL eukaryotic cell transfection, antibody expression and purification, and the titer was tested by supernatant ELISA.

[0052] The specific titer determination method is as follows: 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 ELISA plate, and react overnight at 4°C. 2) Washing: Pour out the liquid in the 96-well ELISA plate, wash the plate three times with PBST buffer, and then pat the liquid in the 96-well ELISA plate dry on filter paper; 3) Blocking: Add 200 µL of 2% BSA (bovine serum albumin) to each well and incubate at 37°C for 2 hours; 4) Wash the plate: repeat step 2); 5) Adding primary antibody: setting the antibody concentrations to 10 ng / mL, 3.33 ng / mL, and 1.11 ng / mL, diluting the antibody according to these three gradient concentrations, adding 100 µL of the diluted antibody (the anti-Ki-67 recombinant monoclonal antibody prepared by the present invention) to each well, placing in an incubator, and incubating at 37°C for 1 hour; 6) Wash the plate: repeat step 2); 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°C for 40 min; 8) Wash the plate: repeat step 2); 9) Color development: Add 100 µL of color development solution to each well, place incubator, incubate at 37°C for 10 min, and add 50 µL of 2 mol / L sulfuric acid solution to terminate the reaction; 10) Determine OD value: Use a microplate reader to measure the absorbance (OD 450) value of each well.

[0053] The results showed that the P0017-VK1 / VH1(R1) ELISA results were as follows Fig.11 As shown, it is positive and has a strong signal, which can well identify the coated Ki-67 protein.

[0054] The P0017-VK1 / VH1(R1) bacterial solution was sent to a sequencing company for sequencing analysis. The sequencing results are as follows: Fig. 9 As shown, the results show that the sequencing peak graph presents obvious, clear and symmetrical peaks, with good resolution and signal-to-noise ratio, and no abnormal peaks or mixed peaks, indicating that the sequencing results are reliable and can be used for subsequent experiments.

[0055] In order to further obtain milligram-level Ki-67 recombinant antibodies for sample verification and characterization, this study used the ExpiCHO-S™ Expression System to perform 60 mL eukaryotic pilot expression and antibody affinity chromatography purification of P0017-VK1 / VH1(R1). The purified recombinant antibodies were analyzed by reducing SDS-PAGE (e.g. Fig.10As shown in the figure, there are clear bands at 25 kDa and 55 kDa without any other bands, which is consistent with the expected target bands.

[0056] Example 4: Identification of recombinant monoclonal Ki-67 In this example, the mouse anti-human Ki-67 recombinant monoclonal antibody prepared according to the method described in Examples 1-3 was subjected to ELISA affinity detection, and the detection process was as follows: To further characterize the affinity of Ki-67 recombinant antibodies, indirect ELISA was used for identification, and then OD 450 nm is the ordinate, and the logarithm of the antibody concentration is the abscissa. The two curves were fitted using the software Origin 8.5 to calculate the OD max / 2 corresponding to the antibody concentration (mol / L), calculate the antibody affinity constant Ka. The result is as follows Fig.12 As shown, A is the concentration of the coated recombinant protein 0.2 μg / mL, and B is the concentration of the coated recombinant protein 0.01 μg / mL According to the affinity constant Ka formula: (Formula 1-1) Wherein, [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, [Ab]2 corresponds to a relatively high coating concentration, and n is the ratio of the two groups of coating concentrations (n>1). 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 10 L / mol, affinity greater than 10 7 L / mol is a high-affinity antibody, indicating that the Ki-67 recombinant antibody prepared by the present invention has a high affinity.

[0057] Embodiment 5: In this example, immunohistochemical detection was performed on the anti-Ki-67 recombinant monoclonal antibody prepared according to the method described in Examples 1-3. The detection process is as follows: Ki-67 is localized in the nucleus, and the proliferation index is closely related to the degree of differentiation, infiltration, metastasis and prognosis of many tumors. Therefore, it is widely used as a marker for various malignant tumors and for tumor research. In order to further verify the ability of Ki-67 recombinant antibodies to recognize Ki-67 protein expression in pathological tissue samples, this example performed immunohistochemical detection on anti-Ki-67 recombinant monoclonal antibodies, and the detection process is as follows: (1) Baking: Place the paraffin sections in a 65°C oven for 2-3 hours to completely melt the paraffin. (2) Dewaxing and hydration: ① Dewaxing with xylene, passing through three tanks of xylene, each time for 5 minutes; ② Hydration: first pass through two tanks of 100% ethanol, each time for 5 minutes, then pass through 95% ethanol for 5 minutes, 70% ethanol for 5 minutes, and finally pass through two tanks of pure water for 5 minutes (machine hydration takes about 50 minutes); (3) Antigen repair: ① Citrate high-pressure antigen repair (100x, pH6.0): Prepare and use immediately. The repair solution must be sufficient to immerse the entire slice. First, boil the tap water in the pressure cooker and then put the repair box (including the high-temperature staining rack) into it. Heat until boiling. First, place the slice on the steam to lubricate it, then put the slice into the repair solution, close the pressure valve, heat until it sprays, start timing for 5 minutes, disconnect the power supply, take out the repair box, leave it at room temperature for 20 minutes, and take out the slice after natural cooling; ② EDTA high-temperature antigen repair (50x, pH9.0): Prepare and use immediately. First, boil the tap water and then put it into the repair box. Heat it to boiling before putting in the slice. Do not close the pressure valve, start timing for 20 minutes, disconnect the power supply, take out the repair box, leave it at room temperature for 20 minutes, and take out the slice after natural cooling; (4) Cleaning: rinse with pure water for 5 min × 2, rinse with PBS buffer for 5 min × 2; (5) Blocking: Add 20-30uL 10% BSA and block in a wet box at 37℃ for 60min, then discard the blocking solution; (6) Primary antibody incubation: add 20-30uL of appropriately diluted primary antibody (anti-Ki-67 recombinant monoclonal antibody prepared by the present invention) and incubate in a wet box at 37°C for 1h; or at 4°C overnight; first add PBS buffer to dilute the primary antibody, and then wash with PBS buffer; (7) Secondary antibody incubation: Wash with PBS buffer for 10 min × 3, add 20-30uL of appropriately diluted secondary antibody (HRP-rabbit anti-mouse IgG), incubate in a wet box at 37°C for 1 h; rinse with PBS for 5 min × 2; (8) DAPI staining of nuclei and sealing of slides: observation under fluorescence, photographing, and storage at 4°C in a dark box if necessary; Some immunohistochemical test results are as follows Fig.13 As shown (Figure A is the immunohistochemical detection of human tonsil by Ki-67 recombinant antibody, and Figure B is the immunohistochemical detection of human liver cancer by Ki-67 recombinant antibody): Ki-67 is localized in the cell nucleus, which is consistent with the expected tissue localization, tissue sublocalization, cell localization and subcellular localization. The signal is strong and is expected to be used for clinical diagnosis.

[0058] In summary, the present invention successfully prepared mouse anti-human Ki-67 recombinant monoclonal antibody, and the antibody has high titer and affinity. The immunohistochemical detection results of Ki-67 recombinant antibody are similar to those of commercial Ki-67 antibody, and in some tissue samples, the immunohistochemical detection positive rate is significantly higher than that of commercial Ki-67 antibody. The antibody can be used for immunohistochemical detection and can provide preliminary data support for clinical pathological diagnosis.

[0059] Finally, it should be emphasized that the above 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 may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-Ki-67 recombinant monoclonal antibody, characterized in that: The anti-Ki-67 recombinant monoclonal antibody comprises 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 according to 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 nucleic acid according to claim 3, characterized in that 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.3 in the anti-Ki-67 recombinant monoclonal antibody; 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.3 in the anti-Ki-67 recombinant monoclonal antibody.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid according to any one of claims 2 to 4.

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

7. A recombinant expression cell, characterized in that: The recombinant expression cell contains the nucleic acid according to any one of claims 2 to 4, or the recombinant expression vector according to claim 5 or 6.

8. A method for preparing an anti-Ki-67 recombinant monoclonal antibody, characterized in that: The preparation method comprises: using the recombinant expression vector as claimed in claim 5 or 6 to transfect cells to obtain recombinant expression cells, culturing the transfected cells to obtain the recombinant expression cells, collecting and purifying the supernatant to obtain the anti-Ki-67 recombinant monoclonal antibody.

9. Use of the nucleic acid according to any one of claims 2 to 4, or the recombinant expression vector according to claim 5 or 6, or the recombinant expression cell according to claim 7 in the preparation of anti-Ki-67 recombinant monoclonal antibodies.

10. Use of the anti-Ki-67 recombinant monoclonal antibody according to claim 1, or the nucleic acid according to any one of claims 2 to 4, or the recombinant expression vector according to claim 5 or 6, or the recombinant expression cell according to claim 7, or the preparation method according to claim 8 in detecting Ki-67 protein molecules, or preparing a device for detecting Ki-67 protein molecules.

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