CN112661842B

CN112661842B - anti-Ki-67 specific monoclonal antibody and application thereof

Info

Publication number: CN112661842B
Application number: CN202110084382.0A
Authority: CN
Inventors: 彭劲武; 刘杨; 闵豆; 程育苗
Original assignee: Suzhou Baidao Medical Technology Co ltd; Central South University
Current assignee: Suzhou Baidao Medical Technology Co ltd; Central South University
Priority date: 2021-01-21
Filing date: 2021-01-21
Publication date: 2022-09-30
Anticipated expiration: 2041-01-21
Also published as: CN112661842A

Abstract

The invention relates to an anti-Ki-67 specific monoclonal antibody, wherein a heavy chain variable region of the monoclonal antibody has a sequence shown as SEQ ID No: 7, and the light chain variable region has the amino acid sequence shown as SEQ ID No: 8. The anti-Ki-67 specific monoclonal antibody encodes the nucleotide sequence of the heavy chain variable region of the anti-Ki-67 specific monoclonal antibody, such as SEQ ID No: 5, the nucleotide sequence of the light chain variable region of the anti-Ki-67 specific monoclonal antibody is shown as SEQ ID No: and 6. The combination of the Ki-67 antibody and the human Ki-67 protein has the characteristics of strong specificity and high detection sensitivity. The antibody is suitable for immunohistochemical detection of malignant tumor tissue specimens of breast cancer, lung cancer, lymphoma, cervical cancer and the like, and can improve the detection accuracy.

Description

anti-Ki-67 specific monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an anti-Ki-67 specific monoclonal antibody and application thereof.

Background

The Ki-67 protein is a nuclear protein related to cell proliferation, is related to ribosomal RNA transcription, and plays a key role in maintaining cell proliferation. The Ki-67 protein is located on the chromosome surface in M phase, is only expressed in cell nucleus in mitosis phase, and does not express Ki-67 in dormant (G0) cells.

The Ki-67 index, i.e., the percentage of cells in the tissue on which Ki-67 stained, represents the growth fraction. For many tumors, the rate of cell proliferation assessed by Ki-67 immunoreactivity correlates with tumor grade and clinical course. In breast cancer, the proliferation index, measured by Ki-67 immunoreactivity, has prognostic and predictive value. In non-Hodgkin's lymphoma, low grade lymphoma with Ki-67 marker index greater than 5% had a worse prognosis than those with Ki-67 index less than 5%. Among gliomas, the Ki-67 index of low-grade astrocytomas ranges from 0% to 5%, while the Ki-67 index most common to anaplastic astrocytomas and glioblastoma is higher than 10%. The sarcoma Ki-67 index is positively correlated with mitotic counts, cellular and histological grade in soft tissue. In some benign tumors, such as meningiomas, a high Ki-67 index is associated with a high recurrence rate. Based on the important application of the Ki-67 index in the aspects of tumor grade evaluation, disease course judgment, prognosis, prediction value and the like, the anti-Ki-67 antibody with high sensitivity and good specificity is obtained and is important for analyzing the Ki-67 index and diagnosing tumors.

Disclosure of Invention

Technical problem to be solved

In view of the problems of the prior art, the invention provides an anti-Ki-67 specific monoclonal antibody which can be widely applied to accurately diagnose various tumors, grade evaluation, course judgment, prognosis, recurrence rate and other predictions.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

in a first aspect, the invention provides an anti-Ki-67 specific monoclonal antibody, wherein the heavy chain variable region of the monoclonal antibody has the amino acid sequence shown in SEQ ID No: 7, and the light chain variable region has an amino acid sequence shown as SEQ ID No: 8, or a pharmaceutically acceptable salt thereof.

In a second aspect, the invention provides an anti-Ki-67 specific monoclonal antibody, wherein the nucleotide sequence of the heavy chain variable region of the anti-Ki-67 specific monoclonal antibody is shown as SEQ ID No: 5, the nucleotide sequence of the variable region of the light chain of the anti-Ki-67 specific monoclonal antibody is shown as SEQ ID No: and 6.

Wherein the subclass of the monoclonal antibody is IgG1 type monoclonal antibody, and the affinity constant is 6 × 10 ^-7 (mol/L). Immunohistochemical experiments show that the anti-Ki-67 monoclonal antibody can recognize Ki-67 protein, including but not limited to recombinant Ki-67 antigen protein and Ki-67 molecule on the surface of tumor cells.

In a third aspect, the invention provides a preparation method of an anti-Ki-67 monoclonal antibody, which comprises the following steps:

s1 immunization: using SEQ ID No:1-4 as immunogen, coupling via KLH or OVA, immunizing different Balb/c mice, taking blood to measure serum antibody titer, and selecting mice with highest antibody titer corresponding to each synthetic polypeptide for cell fusion;

s2 cell fusion: the myeloma cells adopt sp2/0 mouse myeloma-like cells from mice, and are in logarithmic growth phase during fusion; taking mouse spleen lymphocytes and sp2/0 mouse myeloma-like cells for cell fusion, and culturing in HAT culture medium to obtain hybridoma cells;

s3, screening: selecting cell clones within 7-10 days of fusion, performing ELISA test by using Ki-67 antigen, performing immunohistochemical detection by using Ki-67 positive tissues, performing limited dilution on the positive cells, performing ELISA test and IHC detection, and selecting monoclonal stable strains, namely positive hybridoma cell strains 685P3B2, which are all positive and have high positive values, from all diluted samples;

s4 preparation of antibody: preparing mouse ascites by using a cell line of a positive hybridoma cell strain 685P3B2, collecting the ascites, and purifying the ascites by using Protein A/G column affinity chromatography to obtain an anti-Ki-67 specific monoclonal antibody; or,

and (3) culturing and multiplying the positive hybridoma cell strain 685P3B2 by using a serum DMEM culture medium, centrifuging at a low speed, removing the supernatant, transferring the cells to a serum-free culture medium, continuously culturing for 1-2 weeks, collecting cell suspension, centrifuging, taking the supernatant, and purifying by using an affinity chromatography to obtain the anti-Ki-67 specific monoclonal antibody.

Preferably, the synthetic polypeptide used as immunogen in S1 has the amino acid sequence shown in SEQ ID NO 1-4:

SEQ ID NO:1：MWPTRRLVTIKRSGVDGPHF；

SEQ ID NO:2：QALEDLAGFKELFQTRGH；

SEQ ID NO:3：KGIKALKESAKQTPAPAA；

SEQ ID NO:4：EDLAGFKELFQTPG。

in a fourth aspect, the invention provides a preparation method of an anti-Ki-67 specific monoclonal antibody, according to the amino acid sequence SEQ ID No: 7, the amino acid sequence of the light chain variable region SEQ ID No: 8, constructing an expression vector of the Ki-67 antibody in vitro, performing amplification extraction on the expression vector through escherichia coli, removing endotoxin, transfecting Expi-293F cells, continuing culturing after transfection, collecting cell supernatant, and purifying by adopting an affinity chromatography to obtain the anti-Ki-67 specific monoclonal antibody.

In a fifth aspect, the present invention provides a kit or an antibody chip for detecting Ki-67 protein, comprising the anti-Ki-67 specific monoclonal antibody described in any of the above embodiments.

Wherein, the anti-Ki-67 monoclonal antibody can identify recombinant Ki-67 antigen protein and Ki-67 molecules on the surface of tumor cells.

The monoclonal antibody secreted by the positive hybridoma cell strain 685P3B2 can identify tumor cells expressing Ki-67, and can detect various tumor tissues such as breast cancer, pancreatic cancer, melanoma, lymphoma, esophageal cancer, cervical cancer and the like with high Ki-67 protein expression.

The kit containing the anti-Ki-67 specific monoclonal antibody can be applied to detection and screening of Immunohistochemistry (IHC), Western blotting (Western blotting), indirect ELISA, antibody chip preparation and the like, has high specificity and sensitivity, can be used for analyzing Ki-67 indexes, tumor diagnosis, grade evaluation, course judgment, prognosis and prediction of benign recurrence rates such as meningioma, and has the advantages of high affinity, high sensitivity, good specificity and the like.

(III) advantageous effects

(1) The monoclonal antibody secreted by the positive hybridoma 685P3B2 can identify tumor cells expressing Ki-67, and can detect various tumor tissues such as breast cancer, pancreatic cancer, melanoma, lymphoma, esophageal cancer, cervical cancer and the like with high Ki-67 protein expression.

(2) The anti-Ki-67 monoclonal antibody obtained by the invention is an IgG1 antibody, and has extremely strong specificity and sensitivity in combination with Ki-67 protein.

(3) The monoclonal antibody produced by the hybridoma 685P3B2 can be applied to detection and screening such as Immunohistochemistry (IHC), immunoblotting (Western blotting), indirect ELISA, antibody chip preparation and the like, and has high specificity and sensitivity.

(4) The antibody prepared by the cell strain has the specificity and sensitivity basically equivalent to those of a control antibody in the diagnosis of soft tissue sarcoma, prostatic cancer and breast cancer, and has slightly stronger expression intensity than the control antibody. Therefore, the Ki-67 monoclonal antibody of the invention can be widely applied to detection and diagnosis and can accurately diagnose various tumor types.

The 685P3B2 anti-Ki-67 monoclonal antibody has the characteristics of strong specificity and high detection sensitivity when being combined with the human Ki-67 protein. The antibody is suitable for immunohistochemical detection of malignant tumor tissue specimens of breast cancer, lung cancer, lymphoma, cervical cancer and the like, and can improve the detection accuracy.

Drawings

FIG. 1 shows a Western Blot detection result of monoclonal antibodies against Ki-67 positive tissue cells, namely T47D and SH-SY5Y (both recognizing specific bands), wherein the primary antibody is Ki-67 monoclonal antibody 685P3B2 prepared in the examples and used at a concentration of 1. mu.g/mL.

FIG. 2 is a graph comparing the representative results (positive staining signals) of the immunohistochemical detection of Ki-67 monoclonal antibody 685P3B2 and Ki-67 (commercially available Clone MIB-1) monoclonal antibody on tonsils prepared according to the example of the present invention, and the concentration of the primary antibody used was 1. mu.g/mL.

FIG. 3 is a graph comparing the results of immunohistochemical detection (positive staining signals) of breast cancer using the Ki-67 monoclonal antibody 685P3B2 and the Ki-67 (commercially available Clone MIB-1) monoclonal antibody prepared according to the example of the present invention, and the primary antibody was used at a concentration of 1. mu.g/mL.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

The invention is directed to Ki-67 protein molecules, according toThe published sequences were analyzed and, based on structure on the cell membrane, antigenicity, hydropathicity of the constituent amino acids and secondary structure, the sequences of SEQ ID nos: 1-4 as immunogen to immunize Balb/c mouse. The positive hybridoma cell line 685P3B2 capable of efficiently secreting monoclonal antibodies is obtained by fusing, screening and cloning with mouse myeloma-like cells. The hybridoma cell line is used for preparing mouse ascites, and Protein A/G column affinity chromatography is used for purifying the ascites to obtain the anti-Ki-67 monoclonal antibody (or DMEM culture medium of serum is adopted for culture and expansion, then serum-free culture medium is adopted for culture, centrifugation is carried out to obtain supernatant, affinity chromatography is used for purification, and then the anti-Ki-67 monoclonal antibody is prepared). The subclass of the monoclonal antibody is IgG1 type monoclonal antibody determined by ELISA technique, and the affinity constant is 6 x 10 ^-7 (mol/L). Immunohistochemical experiments show that the antibody can specifically recognize Ki-67 protein, including but not limited to recombinant Ki-67 antigen protein, Ki-67 molecules on the surface of tumor cells and the like.

The following explains the embodiments of the present invention with reference to specific examples.

Example 1

Preparation of hybridoma cell strain 685P3B2 secreting anti Ki-67 specific monoclonal antibody:

(1) the following amino acid sequences of the synthetic polypeptide are obtained by chemical synthesis:

SEQ ID NO 1：MWPTRRLVTIKRSGVDGPHF；

SEQ ID NO 2：QALEDLAGFKELFQTRGH；

SEQ ID NO 3：KGIKALKESAKQTPAPAA；

SEQ ID NO 4：EDLAGFKELFQTPG。

(2) mouse immunization: the polypeptides with the sequences shown in SEQ ID NO 1-4 are respectively mixed and emulsified with Freund's complete adjuvant 1:1, different BALB/c mice are respectively immunized by adopting a subcutaneous injection method, and after two weeks, the polypeptides are mixed with Freund's incomplete adjuvant 1:1 for secondary immunization. After twice immunization, blood is taken and subjected to gradient dilution by an ELISA method to determine the serum titer; and determining whether to boost the immunity according to the result. Detecting the serum titer of the mice to reach 1:100000, then performing abdominal cavity boosting immunization (30 mu g) by using the polypeptide (without adjuvant), cutting tail and collecting blood after 3 days, detecting the serum titer, and selecting the mice with the highest antibody titer which are in one-to-one correspondence with the antigen immunization of SEQ ID NO:1-4 for cell fusion.

(3) Hybridoma cell fusion: the myeloma cells used were sp2/0 murine myeloma-like cell lines derived from mice, and were in the logarithmic growth phase at the time of fusion. Taking the spleen of the immunized mouse to prepare lymphocyte single cell suspension. Mixing splenocytes of immunized mouse and SP2/0 mouse myeloma-like cells at a ratio of 4:1 in a 50mL centrifuge tube, adding 50% PEG1500 dropwise, adding incomplete culture medium and the rest stop solution, centrifuging, removing supernatant, adding HAT culture medium (HAT (H-Hypoxanthine Hypoxanthine, A-Aminopterin, T-thymine Thymidine) selective culture medium), suspending, mixing, metering MC to 50mL, packaging in 96-well plate, and standing at 37 deg.C with 5% CO ₂ Culturing in a constant temperature incubator.

(4) Screening and cloning: cell clones were picked at 8 days of fusion and tested by ELISA using the Ki-67 antigen. The cell line number was labeled. The supernatants of the ELISA positive cell lines were subjected to immunohistochemical detection (IHC detection). ELISA and IHC positive well cells were diluted to limit and cultured in 96 well plates. Each well was 100. mu.L, and the cells were cultured in an incubator. Cell supernatants were identified until the full plate results of the 96-well plate were positive by ELISA assay. All the well plates were selected as positive monoclonal stable strains with high positive values, and hybridoma cell 1, 685P3B2, which secreted a specific monoclonal antibody was obtained.

Example 2

The monoclonal antibody is prepared and purified by the following two ways:

the method I comprises the following steps: the cell line 685P3B2 was expanded to about 4X 10 by culturing in DMEM containing 15% serum on a 10cm dish ⁷ At this time, centrifuge at 800rpm for 5 minutes, discard the supernatant and transfer the cells to a 2L spinner flask and add serum free media. Continuing culturing for 1-2 weeks, collecting cell suspension, collecting supernatant, purifying by affinity chromatography, selecting corresponding column material according to antibody subtype, wherein monoclonal antibody 685P3B2 subtype is IgG1, and purifying by protein G. The purified monoclonal antibody was measured for concentration, dispensed (100. mu.L/tube, concentration 1mg/mL), and stored at 4-8 ℃.

Injecting liquid paraffin into the abdominal cavity of the selected mouse to stimulate the immune response of the mouse; inoculating the cell strain 685P3B2 into abdominal cavity of a mouse after one week, extracting ascites after one week, placing the extracted ascites into a centrifuge tube, balancing on a balance, and then placing into a high-speed refrigerated centrifuge for centrifugation; after centrifugation is finished, removing upper-layer grease and sediments with blood filaments at the bottom, filtering the residual liquid by using gauze, adding saturated ammonium sulfate overnight, taking the precipitate, redissolving the precipitate by using PBS, purifying by using an affinity chromatography method, selecting a corresponding column material according to an antibody subtype, wherein the monoclonal antibody 685P3B2 subtype is IgG1, and purifying by using protein G. The purified monoclonal antibody was assayed for concentration, dispensed (100. mu.L/tube, concentration 1mg/mL) and stored at 4-8 ℃.

For ease of description (as distinguished from existing anti-Ki-67 monoclonal antibodies), the anti-Ki-67 specific monoclonal antibody secreted by the hybridoma cells is hereinafter abbreviated as 685P3B2 anti-Ki-67 monoclonal antibody or 685P3B2 anti-Ki-67 monoclonal antibody.

The 685P3B2 anti-Ki-67 monoclonal antibody prepared in the two ways described above was now sequenced:

separating total RNA from hybridoma cells according to the instruction of a reagent TriZol, carrying out reverse transcription on the total RNA into cDNA according to the instruction of a TIANCcript first strand cDNA synthesis kit, amplifying antibody fragments of VH, VL, CH and CL according to specific primers, respectively cloning the amplified antibody fragments into a standard cloning vector, and sequencing.

The sequencing result is as follows: 685P3B2 anti-Ki-67 monoclonal antibody has a heavy chain variable region amino acid sequence consisting of SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the anti-Ki-67 monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No: 6, and a DNA sequence shown in the specification. Correspondingly, the heavy chain variable region amino acid sequence of the anti-Ki-67 monoclonal antibody is SEQ ID No: 7, and the amino acid sequence of the variable region of the light chain of the Ki-67 monoclonal antibody is SEQ ID No: 8.

Example 3

Cell lysates of Ki-67 positive cells T47D (breast cancer cell T47D) and SH-SY5Y (neuroblast) were prepared, and Western Blot detection was performed by adding 685P3B2 anti-Ki-67 monoclonal antibody prepared in example 2 (mode one). The results are shown in FIG. 1, from which it can be seen that the Ki-67 antibody is able to detect a band of Ki-67 protein in lysates of T47D and SH-SY 5Y.

Example 4

Immunohistochemical detection of tonsil tissue samples using 685P3B2 anti-Ki-67 monoclonal antibody as the primary antibody. The method comprises the following steps:

(1) sample preparation: the formalin fixed paraffin embedded section is baked for 1 to 2 hours in an oven at the temperature of 60 ℃ and stored at room temperature for later use.

(2) Dewaxing and hydrating: the paraffin section is placed in fresh dimethylbenzene and soaked for 10 minutes times by 2 times;

after removing the redundant liquid, placing the mixture into absolute ethyl alcohol, and soaking the mixture for 3 minutes and 2 times;

removing redundant liquid, and soaking in 95% ethanol for 3 min;

after removing the redundant liquid, placing the mixture into 85% ethanol, and soaking for 3 minutes;

removing redundant liquid, and soaking in 70% ethanol for 3 min;

purified water was washed 3 min × 2 times.

(3) Antigen retrieval: the high-temperature heat-repairing method of pH9.0EDTA is recommended, and if an automatic repairing instrument is used, the high-temperature repairing can be set at 98 ℃ for 20 minutes, and the slices are naturally cooled to the room temperature. The tissue to be tested (tonsil tissue) was circled with an immunohistochemical pen, and washed with purified water 2 times for 3 minutes each time.

(4) Inactivation of endogenous peroxidase: dropping proper amount of endogenous peroxidase blocker to completely cover the tissue, incubating at room temperature for 10 min, washing with purified water for 2 times (3 min each time), and washing with PBS once.

(5) Primary and blank control antibody incubations: 1-3 drops of 685P3B2 anti-Ki-67 monoclonal antibody and a control commercial MIB-1 monoclonal antibody were added to completely cover the tissue, and after incubation for 1 hour at room temperature, the tissue was washed 3 times with PBS for 5 minutes each.

(6) And (3) secondary antibody incubation: and performing secondary antibody incubation according to the instruction of the DAB staining solution kit of the secondary antibody staining system, washing the film with PBS for 3 times, 5 minutes each time, purifying and washing the film with water for 1 time.

(7) DAB color development: preparing DAB color developing solution according to the instruction of the reagent, dripping proper amount of the prepared DAB color developing solution to completely cover the tissue, stopping dyeing according to color change, and washing with purified water for 3 times.

(8) Hematoxylin lining staining: the sections were back-stained according to the protocol and recommendations of the hematoxylin manufacturer's instructions, washed back to blue with PBS or tap water.

(9) And (3) dehydrating and transparency: sequentially soaking 70%, 85%, 95%, 100% and 100% of gradient alcohol for 3 minutes each time; xylene was clear twice for 10 minutes each.

(10) Sealing: the samples were mounted with neutral gum.

The seal prepared above was observed with a fluorescence microscope, and the result is shown in FIG. 2. The Ki-67 protein is expressed in tonsil tissues in specific cell nuclei, and the 685P3B2 anti-Ki-67 monoclonal antibody detects a significantly stronger Ki-67 signal than the commercially available Ki-67 (commercially available Clone MIB-1) group. Under the same antibody concentration, the dyeing positive signal of the 685P3B2 anti-Ki-67 monoclonal antibody is obviously enhanced compared with the dyeing positive signal of a commercially available Ki-67 (commercially available Clone MIB-1) group, which means that the antibody has higher sensitivity, lower antibody concentration can be used in product design, the production cost is reduced, and the detection is more sensitive and has lower background. The high sensitivity of the antibody enables a stronger signal in IHC staining, the subjective scoring of a pathologist is easier, and the detection and the distinguishing of immune tissues or immune related diseases are more accurate.

Example 5

Immunohistochemical detection using 685P3B2 anti-Ki-67 monoclonal antibody as the primary antibody. Experimental procedures refer to example 4, except that tonsil tissues were changed to breast cancer tissues.

The prepared mounting plate was observed by using a fluorescence microscope, and the result is shown in FIG. 3, the Ki-67 protein is expressed in the specific cell nucleus in the breast cancer tissue, and the signals of the 685P3B2 anti-Ki-67 monoclonal antibody are all obviously stronger than those of the Ki-67 (market Clone MIB-1) group. The Ki-67 (market Clone MIB-1) group has weak staining positive signals and can have the problem of low detection sensitivity in some tissues with low malignancy degree or early cancer, and the antibody of the invention has the characteristics of good specificity, strong positive signals and the like, so that the antibody can be scored more easily in IHC staining and can be used for detecting and distinguishing cancers more accurately.

Example 6

The subtype of the 685P3B2 anti-Ki-67 monoclonal antibody is detected by an ELISA method. The result showed that 685P3B2 anti-Ki-67 monoclonal antibody was of the subtype IgG 1.

Example 7

685P3B2 anti Ki-67 monoclonal antibody affinity assay, the method is as follows:

(1) the polypeptide antigen as Ki-67 of the sequences SEQ ID NO 1-4 was removed from 4 ℃. Return to room temperature. Different polypeptide molarities were prepared and 100. mu.L/well was added to a 96-well plate.

(2) 685P3B2 antibody was diluted to an initial mass concentration of 0.125. mu.g/mL, antibody molecular weight of 150kD, and molar concentration of approximately 0.83 nmol/L.

(3) The diluted 685P3B2 anti-Ki-67 monoclonal antibody was added to a 96-well sample containing the polypeptide, 100. mu.L/well, and mixed with serial dilutions of the antigen by pipetting. The plate-sealing membrane was covered and incubated at 37 ℃ for 1 hour to allow the reaction to equilibrate.

(4) The ELISA plate was removed from 37 deg.C, the liquid was emptied, rinsed 3 times, and water was tapped off.

(5) Diluted HRP-labeled goat anti-mouse IgG was added to the ELISA plate at 100. mu.L/well and incubated at 37 ℃ for 1 hour.

(6) The ELISA plate was removed from 37 ℃ and the liquid was emptied, rinsed 5 times and drained.

(7) TMB developing solution was added to 100. mu.L of each well, and the reaction was carried out at room temperature for 6 minutes.

(8) 50 μ L of 2M H per well ₂ SO ₄ The color development was terminated.

(9) Reading OD value at 450nm on enzyme labeling instrument, collating data and analyzing result

The results showed that the 685P3B2 anti-Ki-67 monoclonal antibody affinity constant at a polypeptide molar concentration of 0.82. mu. mol/L was: 6X 10 ^-7 (mol/L)。

Example 8

The amino acid sequence and the DNA sequence of the 685P3B2 anti-Ki-67 monoclonal antibody disclosed by the invention are shown in SEQ ID NO: 5-8, constructing an expression vector of the Ki-67 antibody in vitro. The expression vector is amplified and extracted by escherichia coli, endotoxin is removed, Expi-293F cells are transfected, culture is continued after transfection, cell supernatant is collected, and the anti-Ki-67 specific monoclonal antibody is obtained by adopting an affinity chromatography purification method.

The specific method comprises the following steps:

(1) construction of an expression vector: the light chain variable region (VL) sequence (SEQ ID NO: 8) of the anti-Ki-67 specific monoclonal antibody was constructed to the kappa chain constant region (SEQ ID NO:9) of the murine antibody light chain, and the heavy chain variable region (VH) sequence (SEQ ID NO: 7) of the anti-Ki-67 specific monoclonal antibody was constructed to the constant region (SEQ ID NO:10, murine IgG1) of the murine antibody heavy chain. Adding optimized signal peptide into the N end of the heavy chain/light chain amino acid of the recombinant antibody for secretory expression, performing codon optimization on the amino acid of the recombinant antibody, adding a Kozak sequence GCCGCCACC into the 5' end of the nucleotide, adding EcoRI/HindIII enzyme cutting sites of pcDNA3.4 into the two ends of the nucleotide, and connecting the synthesized gene to a pcDNA3.4 vector after enzyme cutting to obtain an expression vector.

(2) Transfection: the expression vector is amplified and extracted by escherichia coli, endotoxin is removed, and the expression vector is amplified and extracted by the plasmids: medium =1 μ g/ml transfected Expi-293F cells. The Transfection reagent used was Expifactamine 293Transfection Kit (Theromfisher, Lot #: A14524) with a cell density of 25 x 10 at Transfection ⁵ cells/ml, expression enhancers Enhancer1 and Enhancer2 were added 18h after transfection, and cell supernatants were collected 5 days after transfection.

(3) Protein A purification: centrifuging the supernatant at 10000rpm/min at 4 deg.C for 30min to remove cell debris, equilibrating the Protein A column with equilibration solution (0.02MPB, 0.15M NaCl, pH7.0) for 10 column volumes, flowing the supernatant through the column at a rate of 2ml/min, washing the combined column with equilibration solution for 5 column volumes, adding eluent (0.02M PB, 0.15M NaCl, pH3.0) for elution, and dropping the eluent into a collection tube containing neutralization solution (1M Tris, pH 9.0). The protein eluate was collected, concentrated by ultrafiltration using an ultrafiltration tube (Millipore UFC903096)4000G and buffer was replaced with PBS (HyClone SH30256.01), and the eluate was stored at-20 ℃ after detection by SDS-PAGE. Removing endotoxin, filtering for sterilization, and detecting purity by SDS-PAGE electrophoresis.

The anti-Ki-67 specific monoclonal antibody prepared by the method is added into a lysate of a breast cancer cell T47D to carry out Western Blot detection, and a Ki-67 protein band can be detected by the Ki-67 antibody, which is the same as the case shown in FIG. 1.

In addition to the preparation method, when the 685P3B2 anti-Ki-67 monoclonal antibody is produced in a large scale, a plasmid vector can be constructed according to the antibody coding sequence disclosed by the invention, the plasmid vector is transferred to cells to obtain a stable expression cell strain, fermentation culture is carried out by using a fermentation tank to obtain the cell supernatant or lysate of the stable transfer strain, and the cell supernatant or lysate is purified by adopting an affinity chromatography method to produce and prepare the 685P3B2 anti-Ki-67 monoclonal antibody.

Finally, it should be noted that: it is also within the scope of the present claims to be covered by those skilled in the art to produce 685P3B2 anti Ki-67 monoclonal antibody based on the amino acid sequence of 685P3B2 anti Ki-67 monoclonal antibody disclosed herein and the DNA sequence encoding the same (SEQ ID NO: 5-8) by any existing method or methods that may be possible in the future. Similarly, the commercial application of 685P3B2 anti-Ki-67 monoclonal antibody in any form, including but not limited to kits, antibody chips, and the like, should be covered by the scope of the present claims.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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<400> 8

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

1 5 10 15

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

20 25 30

Gly Gln Ser Asn Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Val His Trp Pro Thr Phe Gly Gly Gly Thr Lys Leu

100 105

<210> 9

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 9

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 10

<211> 324

<212> PRT

<213> Artificial Sequence

<400> 10

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

Claims

1. An anti-Ki-67 specific monoclonal antibody, which is characterized in that the heavy chain variable region is SEQ ID No: 7, and the variable region of the light chain is an amino acid sequence shown as SEQ ID No: 8.

2. An anti-Ki-67 specific monoclonal antibody, which is characterized in that the nucleotide sequence of the heavy chain variable region of the anti-Ki-67 specific monoclonal antibody is shown as SEQ ID No: 5, and the nucleotide sequence of the light chain variable region of the anti-Ki-67 specific monoclonal antibody is shown as SEQ ID No: and 6.

3. The anti-Ki-67 specific monoclonal antibody according to claim 1 or 2, characterized in that the anti-Ki-67 specific monoclonal antibody is a mouse IgG1 subtype monoclonal antibody.

4. The preparation method of the anti-Ki-67 specific monoclonal antibody is characterized in that the amino acid sequence of the heavy chain variable region of the anti-Ki-67 specific monoclonal antibody is SEQ ID No: 7, amino acid sequence of light chain variable region of anti Ki-67 specific monoclonal antibody SEQ ID No: 8, constructing an expression vector of the Ki-67 antibody in vitro, performing amplification extraction on the expression vector through escherichia coli, removing endotoxin, transfecting Expi-293F cells, continuing culturing after transfection, collecting cell supernatant, and purifying by adopting an affinity chromatography to obtain the anti-Ki-67 specific monoclonal antibody.

5. A kit or an antibody chip for detecting Ki-67 protein, which comprises the anti-Ki-67 specific monoclonal antibody according to any one of claims 1 to 3 or the anti-Ki-67 specific monoclonal antibody prepared by the preparation method according to claim 4.