阅读说明：本技术 一种抗cd23特异性单克隆抗体及其应用 (anti-CD 23 specific monoclonal antibody and application thereof ) 是由 彭劲武 刘杨 闵豆 程育苗 于 2021-01-21 设计创作，主要内容包括：本发明涉及一种抗CD23特异性单克隆抗体,其重链可变区具有如SEQ ID No：6所示的氨基酸序列,其轻链可变区具有如SEQ ID No：7所示的氨基酸序列。该抗CD23特异性单克隆抗体,编码所述抗CD23特异性单克隆抗体的重链可变区的核苷酸序列如SEQ ID No：4所示,编码所述抗CD23特异性单克隆抗体的轻链可变区的核苷酸序列如SEQ ID No：5所示。本发明的抗CD23特异性单克隆抗体能准确识别淋巴造血系统中CD23分子,在慢性淋巴细胞白血病、纵隔大B细胞淋巴瘤和系统性红斑狼疮等疾病中呈阳性高表达。因此本发明抗CD23特异性单克隆抗体可用于准确诊断各种淋巴造血疾病,以提高诊断检测的准确度。(The invention relates to an anti-CD 23 specific monoclonal antibody, wherein the heavy chain variable region thereof has the amino acid sequence shown in SEQ ID No: 6, and the light chain variable region has the amino acid sequence shown as SEQ ID No: 7. The anti-CD 23 specific monoclonal antibody has the nucleotide sequence of the heavy chain variable region of the anti-CD 23 specific monoclonal antibody as shown in SEQ ID No: 4, the nucleotide sequence of the light chain variable region of the monoclonal antibody for encoding the anti-CD 23 specificity is shown as SEQ ID No: 5, respectively. The anti-CD 23 specific monoclonal antibody can accurately identify CD23 molecules in a lymphohematopoietic system, and shows positive high expression in diseases such as chronic lymphocytic leukemia, mediastinal large B cell lymphoma, systemic lupus erythematosus and the like. Therefore, the anti-CD 23 specific monoclonal antibody of the invention can be used for accurately diagnosing various lymphohematopoietic diseases so as to improve the accuracy of diagnosis and detection.)

1. An anti-CD 23 specific monoclonal antibody, wherein the heavy chain variable region has the amino acid sequence as shown in SEQ ID No: 6, and the light chain variable region has the amino acid sequence shown as SEQ ID No: 7.

2. An anti-CD 23 specific monoclonal antibody, wherein the nucleotide sequence of the heavy chain variable region encoding the anti-CD 23 specific monoclonal antibody is shown as SEQ ID No: 4, the nucleotide sequence of the light chain variable region of the monoclonal antibody for encoding the anti-CD 23 specificity is shown as SEQ ID No: 5, respectively.

3. The anti-CD 23-specific monoclonal antibody according to claim 1 or 2, wherein the anti-CD 23 monoclonal antibody is a mouse IgG1 subtype monoclonal antibody.

4. A method for preparing an anti-CD 23 specific monoclonal antibody, which is characterized by comprising the following steps:

s1 immunization: using SEQ ID No:1-3 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 are sp2/0 mouse myeloma-like cells of mouse origin; 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 CD23 antigen, performing immunohistochemical detection by using CD23 positive tissue, performing limited dilution on positive cells, performing ELISA test and IHC detection, and selecting monoclonal stable strains which are positive and have high positive values and are all diluted samples as positive hybridoma cell strains;

s4 preparation of antibody: preparing mouse ascites by using the cell line of the positive hybridoma cell line, collecting the ascites, and purifying the ascites by using Protein A/G column affinity chromatography to obtain an anti-CD 23 specific monoclonal antibody; alternatively, the first and second electrodes may be,

and (2) culturing and expanding the cell line of the positive hybridoma cell strain by using a DMEM medium of serum, centrifuging at a low speed, removing a supernatant, transferring the cells to a serum-free medium, continuously culturing for 1-2 weeks, collecting cell suspension, centrifuging, taking the supernatant, and purifying by using an affinity chromatography method to obtain the anti-CD 23 specific monoclonal antibody.

5. A method for preparing an anti-CD 23 specific monoclonal antibody, which is characterized in that the amino acid sequence of the heavy chain variable region of the anti-CD 23 specific monoclonal antibody is SEQ ID No: 6, the amino acid sequence of the light chain variable region of the anti-CD 23 specific monoclonal antibody SEQ ID No: 7, constructing an expression vector of the CD23 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-CD 23 specific monoclonal antibody.

6. A kit or antibody chip comprising the anti-CD 23-specific monoclonal antibody of any one of claims 1-3 or the anti-CD 23-specific monoclonal antibody produced by the production method of any one of claims 4-5.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an anti-CD 23 specific monoclonal antibody and application thereof.

Background

CD23 is a type II transmembrane glycoprotein with a relative molecular weight of 45kDa, a low affinity IgE receptor, and the only Fc receptor not belonging to the Ig superfamily. CD23 is expressed primarily in immune cells and is an important molecule for B cell activation and growth. It can also be expressed on the surface of T lymphocytes, eosinophils, monocytes, dendritic cells, platelets, intestinal epithelial cells, and bone marrow stromal cells. CD23 is expressed on the surface of these cells and performs a variety of functions such as modulation of IgE synthesis, cell-to-cell adhesion, antigen presentation, early T cell differentiation, histamine release by mast cells and basophils, immune attack against parasites, prevention of germinal center B cell apoptosis, modulation of inflammatory responses, modulation of HIV1 expansion, and the like.

The research shows that the CD23 can be used as an index for diagnosis and prognosis of neoplastic diseases and a potential therapeutic target. In lymphohematopoietic diseases, CD23 marks thymic medullary B cells, and the primary mediastinal (thymus) large B cell lymphoma 60-85% expresses CD23 according to literature reports, while the expression rate in non-mediastinal diffuse large B cell lymphoma and extranodal diffuse large B cell lymphoma is lower, and the lymphohematopoietic disease has higher sensitivity, specificity and positive predictive value. In addition, some studies have found that CD23 is associated with a variety of diseases including allergic diseases, autoimmune diseases, leukemia, and glomerular diseases. The positive rate of the peripheral blood lymphocyte CD23 of the diseases is obviously increased and is positively correlated with the severity of the diseases, the positive rate of the CD23 is obviously reduced after effective treatment, and the researches provide a new way for exploring pathogenesis and treatment of the diseases. The CD 23-based detection index is important to be applied to various malignant tumors, metastasis, prognosis and the like, so that the anti-CD 23 antibody with high sensitivity and strong specificity is of great significance.

Disclosure of Invention

Technical problem to be solved

In view of the problems of the prior art, the invention provides an anti-CD 23 specific monoclonal antibody, which can be widely applied to diagnosis of various lymphohematopoietic diseases, has extremely high specificity and sensitivity, and is beneficial to accurate diagnosis.

The anti-CD 23 specific monoclonal antibody provided by the invention is produced by a mouse hybridoma cell strain, and is found by immunohistochemical detection of various tissues, the antibody can well identify the occurrence of tumors in a lymphohematopoietic system, and can be used for immunological diagnosis of diseases such as the lymphohematopoietic system.

(II) technical scheme

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

in a first aspect, the present invention provides an anti-CD 23 specific monoclonal antibody, wherein the heavy chain variable region has the amino acid sequence as shown in SEQ ID No: 6, and the light chain variable region has the amino acid sequence shown as SEQ ID No: 7.

In a second aspect, the present invention provides an anti-CD 23 specific monoclonal antibody, wherein the nucleotide sequence of the heavy chain variable region encoding the anti-CD 23 specific monoclonal antibody is as shown in SEQ ID No: 4, the nucleotide sequence of the light chain variable region of the monoclonal antibody for encoding the anti-CD 23 specificity is shown as SEQ ID No: 5, respectively.

Wherein the subclass of the anti-CD 23 monoclonal antibody is mouse IgG1 monoclonal antibody, and the affinity constant is 7 × 10^-7(mol/L). Immunohistochemical experiments show that the antibody can specifically recognize CD23 protein, including but not limited to recombinant CD23 antigen protein and CD23 molecule in lymphohematopoietic system.

In a third aspect, the present invention provides a method for preparing an anti-CD 23 monoclonal antibody, comprising:

s1 immunization: using SEQ ID No:1-3 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 are sp2/0 mouse myeloma-like cells of mouse origin; 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 CD23 antigen, performing immunohistochemical detection by using CD23 positive tissue, performing limited dilution on positive cells, performing ELISA test and IHC test, and selecting a monoclonal stable strain which is a hybridoma cell strain 365A2D1 and is positive in all diluted samples and high in positive value;

s4 preparation of antibody: preparing mouse ascites by using a cell line of a hybridoma cell strain 365A2D1, collecting the ascites, and purifying the ascites by using Protein A/G column affinity chromatography to obtain an anti-CD 23 specific monoclonal antibody; alternatively, the first and second electrodes may be,

and (3) culturing and expanding the hybridoma cell strain 365A2D1 by adopting a serum DMEM medium, centrifuging at a low speed, removing a supernatant, transferring cells to a serum-free medium, continuously culturing for 1-2 weeks, collecting cell suspension, centrifuging, taking the supernatant, and purifying by using an affinity chromatography method to obtain the anti-CD 23 specific monoclonal antibody.

The anti-CD 23 monoclonal antibody was secreted by the mouse hybridoma cell line 365A2D 1.

The preparation method also comprises the steps of monoclonal antibody subtype identification and affinity constant determination: the obtained anti-CD 23 specific monoclonal antibody subtype has IgG1 type monoclonal antibody subclass determined by ELISA and 7 × 10 affinity constant^-7(mol/L)。

The result of immunohistochemical experimental detection shows that the antibody has the function of specifically recognizing CD23 protein molecules in malignant tumor tissues.

Preferably, the synthetic polypeptide used as immunogen in S1 is the amino acid sequence shown in SEQ ID NO1-3, SEQ ID NO1 is DPDGRLPTPSAPLHS; 2 is CVMMRGSGRWNDAF; SEQ ID NO 3 is SQELEELRAEQQRLK.

In a fourth aspect, the present invention provides a method for preparing an anti-CD 23 specific monoclonal antibody, wherein the heavy chain variable region of the anti-CD 23 specific monoclonal antibody has the amino acid sequence of SEQ ID No: 6, the amino acid sequence of the light chain variable region SEQ ID No: 7, constructing an expression vector of the CD23 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 an affinity chromatography method to obtain the anti-CD 23 specific monoclonal antibody.

In a fifth aspect, the present invention provides a kit or an antibody chip comprising the anti-CD 23-specific monoclonal antibody according to any one of the above embodiments.

Preferably, the anti-CD 23 monoclonal antibody is used for detecting the expression of CD23 in lymphohematopoietic system and normal tissue cells, and the detection method comprises one or more of the following methods: immunohistochemistry, immunoblotting, and enzyme-linked adsorption assays.

Preferably, the anti-CD 23 monoclonal antibody is used in an immunohistochemical pathological diagnostic agent.

(III) advantageous effects

The anti-CD 23 monoclonal antibody is secreted by a cell line of a positive hybridoma cell 365A2D1, is an IgG1 antibody, can accurately identify the expression of CD23 in a lymphohematopoietic system, and shows positive high expression in diseases such as chronic lymphocytic leukemia, mediastinal large B cell lymphoma, systemic lupus erythematosus and the like. The monoclonal antibody produced by the hybridoma 365A2D1 cell line obtained by the invention can be applied to detection and screening of Immunohistochemistry (IHC), indirect ELISA, Western blotting, antibody chip preparation and the like, and has strong specificity and high sensitivity.

Drawings

FIG. 1 is a comparison graph of representative results (staining positive signals) of immunohistochemical detection of CD23 monoclonal antibody 365A2D1 and commercially available CD23 monoclonal antibody (currently the most commonly used commercial CD23 antibody) on tonsil tissue samples prepared according to the example of the present invention, wherein the concentration of primary antibody used is 1 μ g/mL.

FIG. 2 is a graph comparing the results of immunohistochemical detection (positive staining signals) of the CD23 monoclonal antibody 365A2D1 and the commercially available CD23 monoclonal antibody (currently most commonly used commercial CD23 antibody) on colon cancer tissue samples prepared in the example of the present invention, wherein the concentrations of the primary antibody used were 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 CD23 protein molecule is analyzed according to published sequences, and according to the structure on a cell membrane, the antigenicity, the hydrophilicity and hydrophobicity of the constituent amino acids and the secondary structure, the protein of the invention selects the amino acid sequence shown in SEQ ID No:1-3 as immunogen to immunize Balb/c mouse. The positive hybridoma cell line 365A2D1 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-CD 23 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-CD 23 monoclonal antibody is prepared). The subclass of the monoclonal antibody is IgG1 type monoclonal antibody determined by ELISA technique, and the affinity constant is 7 × 10^-7(mol/L). Immunohistochemical experiments show that the antibody can specifically recognize CD23 protein, including but not limited to recombinant CD23 antigen protein and CD23 molecule in lymphohematopoietic system.

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

Example 1

Preparation of hybridoma cell line 365A2D1 secreting anti-CD 23 specific monoclonal antibody:

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

SEQ ID NO 1：DPDGRLPTPSAPLHS；

SEQ ID NO 2：CVMMRGSGRWNDAF；

SEQ ID NO 3：SQELEELRAEQQRLK。

(2) mouse immunization: mixing and emulsifying the polypeptides with the sequence shown in SEQ ID NO1-3 with Freund's complete adjuvant 1:1, respectively, immunizing different BALB/c mice by subcutaneous injection, mixing and emulsifying the polypeptides with Freund's incomplete adjuvant 1:1 at an interval of two weeks, and performing 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, selecting the mice with the highest antibody titer corresponding to the antigen immunization of SEQ ID No:1-3, and preparing for cell fusion.

(3) Hybridoma cell fusion: the myeloma cells were derived from mouse sp2/0 mouse myeloma-like cell line and were in 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) to suspend, mixing, metering MC culture medium to 50mL, packaging in 96-well plate, standing at 37 deg.C and 5% CO₂Culturing in a constant temperature incubator.

(4) Screening and cloning: cell clones were picked at 8 days of fusion and tested by ELISA using CD23 antigen. The cell line number was labeled. The supernatants of the ELISA-positive cell lines were subjected to immunohistochemical detection (i.e., IHC detection). ELISA and IHC positive well cells were diluted in a limiting manner 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 secreting a specific monoclonal antibody, 365A2D1, was obtained.

Example 2

The preparation and purification of the monoclonal antibody specific to the CD23 adopt the following two modes:

the first method is as follows: cell line 365A2D1 was expanded to approximately 4X 10 by culturing in 10cm dishes in DMEM medium containing 15% serum⁷In one occasion, centrifuging at 1200rpm for 6 minutes, removing the supernatant, transferring the cells into A2L spinner flask, adding a serum-free culture medium for continuous culture for 2 weeks, collecting cell suspension, centrifuging, taking the supernatant, purifying the supernatant by an affinity chromatography method, selecting a corresponding column material according to an antibody subtype, wherein the monoclonal antibody 365A2D1 subtype is IgG1, and purifying by adopting proteinG. Measuring the concentration of the purified monoclonal antibody, subpackaging (100 μ L/tube, concentration of 1mg/mL), and storing in a refrigerator at 4-8 deg.C.

Injecting liquid paraffin into the abdominal cavity of the selected mouse to stimulate the immune response of the mouse; inoculating the cell strain 365A2D1 into the abdominal cavity of the mouse after one week, extracting ascites after one week, putting the extracted ascites into a centrifuge tube, balancing on a balance, and then putting into a high-speed refrigerated centrifuge for centrifugation; after the 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 by using PBS, purifying by using an affinity chromatography, selecting a corresponding column material according to the antibody subtype, wherein the monoclonal antibody 365A2D1 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 convenience of description (to distinguish from the existing anti-CD 23 monoclonal antibody), the anti-CD 23 specific monoclonal antibody secreted from hybridoma cells is hereinafter abbreviated as 365A2D1 anti-CD 23 monoclonal antibody or 365A2D1 anti-CD 23 monoclonal antibody.

The 365A2D1 anti-CD 23 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: the amino acid sequence of the heavy chain variable region of the 365A2D1 anti-CD 23 monoclonal antibody consists of SEQ ID No: 4, and the light chain variable region amino acid sequence of the anti-CD 23 monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No: 5 is encoded by the DNA sequence shown in the specification. Correspondingly, the heavy chain variable region amino acid sequence of the anti-CD 23 monoclonal antibody is SEQ ID No: 6, and the amino acid sequence of the light chain variable region of the CD23 monoclonal antibody is shown in SEQ ID No: 7.

Example 3

The subtype of 365A2D1 antibody was detected by ELISA. The results showed that the subtype of the 365A2D1 antibody was IgG 1.

Example 4

Immunohistochemical detection of tonsil tissue samples using 365A2D1 anti-CD 23 monoclonal antibody as the primary antibody. The method comprises the following steps:

(1) sample preparation: the formalin-fixed paraffin-embedded sections were baked in a 60 ℃ incubator for 2 hours and stored at room temperature for future use.

(2) Dewaxing: paraffin slices are soaked in fresh dimethylbenzene for 2 times for dewaxing, and each time is 10 min.

(3) Hydration of the slices: sequentially soaking in anhydrous ethanol (2 times, 3min each time), 95% ethanol (3min), 85% ethanol (3min), and 70% ethanol for 5min for hydration, washing with purified water for 2 times, 3min each time.

(4) Antigen retrieval: repairing with high temperature heat repairing method for 3min (98 deg.C for 20min if using automatic repairing instrument), naturally cooling to room temperature, looping the tissue to be tested (lung adenocarcinoma tissue in this embodiment) with immunohistochemical pen, washing with purified water for 2 times, 3min each time.

(5) Inactivation of endogenous peroxidase: dropping appropriate amount of endogenous peroxidase blocker to completely cover the tissue, incubating at room temperature for 10min, washing with purified water for 2 times (3min each time), and washing with PBST for 1 time.

(6) Primary and blank control antibody incubations: 100ul of 365A2D1 anti-CD 23 monoclonal antibody and a control commercial CD23 antibody were added to completely cover the tissue, and the tissue was incubated at 37 ℃ for 1h and washed 3 times with PBST (phosphate buffered saline containing Tween-20) for 5min each.

(7) And (3) secondary antibody incubation: and (4) performing secondary antibody incubation according to the instruction of the DAB staining solution kit of the secondary antibody staining system, after the incubation is finished, washing the PBST by using the washing sheet for 3 times, 5min each time, and washing by using purified water for 1 time.

(8) DAB color development: preparing DAB color developing solution according to the DAB staining solution kit specification, dripping a proper amount of the prepared DAB color developing solution until the tissue is completely covered, stopping staining when the color is not deepened, and washing with purified water for 3 times.

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

(10) And (3) dehydrating and transparency: soaking in 70%, 85%, 95%, 100%, 100% gradient alcohol for 3min each time; 2 times, 5min each time, xylene was clear.

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

As a result of observing the above-prepared mounting plate using a fluorescence microscope, as shown in fig. 1, CD23 protein was expressed in a specific cell membrane in tonsil tissues, and the detection signal (fluorescence intensity) of the anti-CD 23 monoclonal antibody cloned from 365A2D1 was significantly stronger than that of the commercially available CD23 group. Under the same antibody concentration (1 mu g/mL), the dyeing positive signal of the 365A2D1 anti-CD 23 monoclonal antibody is obviously enhanced compared with the dyeing positive signal of a commercially available CD23 group, which shows that the antibody has higher sensitivity, lower antibody concentration can be used in product design, the production cost is reduced, and the background is reduced under the condition of ensuring the sensitivity. The pathological doctor can judge more conveniently according to the CD23 detection result and experience, and can detect and distinguish immune tissues or immune related diseases more accurately.

Example 5

Immunohistochemical detection using the 365A2D1 anti-CD 23 monoclonal antibody as the primary antibody. Experimental procedures reference example 5 except that lung adenocarcinoma tissue was changed to "colon cancer tissue".

The prepared mounting pieces were observed by a fluorescence microscope, and as a result, as shown in fig. 2, the CD23 protein was expressed in a specific cell membrane in colon cancer tissues, and the signals (fluorescence intensities) of the 365A2D1 anti-CD 23 monoclonal antibodies were all significantly stronger than those of the commercially available CD23 group. The commercial CD23 group has weak positive staining signals, and has the problem of low detection sensitivity in some early cancer tissues. The 365A2D1 anti-CD 23 monoclonal antibody has the characteristics of good specificity, strong positive signal and the like, so that the evaluation in IHC staining is easier, and the detection and the cancer distinguishing are more accurate, particularly the detection of some early cancer tissues.

Example 6

365A2D1 anti-CD 23 monoclonal antibody affinity determination, the method is as follows:

(1) the polypeptide antigen of CD23 as shown in SEQ ID NO1-3 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) 365A2D1 antibody was diluted to an initial mass concentration of 0.125. mu.g/mL, an antibody molecular weight of 150kD, and a molar concentration of approximately 0.83 nmol/L.

(3) Adding the diluted 365A2D1 anti-CD 23 monoclonal antibody to a 96-well enzyme label plate with polypeptide according to 100 mu L/well, covering a sealing plate membrane, and incubating at the constant temperature of 37 ℃ for 1h to ensure that the reaction reaches the balance.

(4) Taking out the enzyme label plate from the incubation chamber at 37 ℃, discarding liquid, washing with purified water for 5 times, and patting to dry.

(5) According to the instructions of the secondary antibody, diluted HRP-labeled goat anti-mouse IgG is added into the ELISA plate at the rate of 100. mu.L/hole, and the reaction is incubated at the constant temperature of 37 ℃ for 1h to reach the equilibrium.

(6) Taking out the enzyme label plate from the incubation chamber at 37 ℃, discarding liquid, washing with purified water for 5 times, and patting to dry.

(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 well₂SO₄The color development was terminated.

(9) And (4) reading the OD value at 450nm on a microplate reader, collating the data and analyzing the result.

The results show that at a polypeptide molar concentration of 0.56. mu. mol/L, the affinity constant of the 365A2D1 anti-CD 23 monoclonal antibody is: 7X 10^-7(mol/L)。

Example 7

The amino acid sequence and the DNA sequence of the 365A2D1 anti-CD 23 monoclonal antibody disclosed by the invention are shown in SEQ ID NO: 4-7, constructing an expression vector of the CD23 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 monoclonal antibody with CD23 resistance specificity is obtained after purification by adopting an affinity chromatography.

The specific method comprises the following steps:

(1) construction of an expression vector: the light chain variable region (VL) sequence (SEQ ID NO: 7) of the anti-CD 23-specific monoclonal antibody was constructed to the kappa chain constant region (SEQ ID NO:8) of the murine antibody light chain, and the heavy chain variable region (VH) sequence (SEQ ID NO: 6) of the anti-CD 23-specific monoclonal antibody was constructed to the constant region of the murine antibody heavy chain, preferably murine IgG1(SEQ ID NO: 9). 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: Expi-293F cells were transfected at 1 μ g/ml medium. The Transfection reagent used was Expifactamine 293Transfection Kit (Theromfisher, Lot #: A14524) with a cell density of 25 x 10 at Transfection⁵cells/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 monoclonal antibody with the specificity of anti-CD 23, which is prepared by the method, is added into cell lysate of tonsil tissues for Western Blot detection, so that an obvious CD23 protein band can be detected.

In addition to the above preparation method, when the 365A2D1 anti-CD 23 monoclonal antibody of the invention 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, the stable expression cell strain is fermented and cultured by a fermentation tank to obtain cell supernatant or lysate of the stable expression cell strain, and the cell supernatant or lysate is purified by affinity chromatography (corresponding column materials, such as protein A/G columns, are selected according to antibody subtypes) to produce and prepare the 365A2D1 anti-CD 23 monoclonal antibody.

Finally, it should be noted that: the 365A2D1 anti-CD 23 monoclonal antibody can be prepared by any existing method or a method possible in the future on the basis of the amino acid sequence of the 365A2D1 anti-CD 23 monoclonal antibody disclosed in the application and the DNA sequence coding the amino acid sequence by the skilled person in the art, and the method is also covered by the protection scope of the patent claims. Similarly, any type of commercial applications of the 365A2D1 anti-CD 23 monoclonal antibody, including but not limited to kits, antibody chips, and the like, are also intended to be covered by the scope of the present invention.

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.

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cctggcaagg gcctggagtg ggtggccagg atcaggagca agagcaacaa ctacgagcct 180

tactacgccg acagcgtgaa ggacaggttc accatcagca gggacgacag ccagagcatc 240

ctgtacctgc agatgaacaa cctgaagacc gaggacaccg ccatgtacta ctgcgtgagg 300

gagtactacg acgccatgga ctactggggc cagggcacca gcgtgaccgt gagcagc 357

<210> 5

<211> 336

<212> DNA

<213> Artificial Sequence

<400> 5

gagatcgtga tgacccaggc cgcccctagc gtgagcgtga cccctggcga gagcgtgagc 60

atcagctgca ggagcagcca gagcctgctg cacagcaacg gcaacaccta cctgtactgg 120

ttcctgcaga ggcctggcca gagccctcac ctgctgatca gcagggtgag caacctggcc 180

agcggcgtgc ctgacaggtt cagcggcagc ggcagcggca ccgccttcac cctgaggatc 240

agcagggtgg aggccgagga cgtgggcgtg tactactgca tgcagggcct ggagttccct 300

tggaccttcg gcggcggcac caagctggag atcaag 336

<210> 6

<211> 119

<212> PRT

<213> Artificial Sequence

<400> 6

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

1 5 10 15

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

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Glu Pro Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Arg Glu Tyr Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 7

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 7

Glu Ile Val Met Thr Gln Ala Ala Pro Ser Val Ser Val Thr Pro Gly

1 5 10 15

Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro His Leu Leu Ile Ser Arg Val Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Leu Glu Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 8

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 8

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> 9

<211> 324

<212> PRT

<213> Artificial Sequence

<400> 9

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