CN106554418B

CN106554418B - Anthrax toxin receptor 2 monoclonal antibody, preparation method and application

Info

Publication number: CN106554418B
Application number: CN201610919910.9A
Authority: CN
Inventors: 易维京; 卞修武; 崔有宏; 蔡瑞丽
Original assignee: First Affiliated Hospital of TMMU
Current assignee: First Affiliated Hospital of TMMU
Priority date: 2016-10-21
Filing date: 2016-10-21
Publication date: 2020-06-23
Anticipated expiration: 2036-10-21
Also published as: CN106554418A

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to an anthrax toxin receptor 2 monoclonal antibody, a preparation method and application thereof. The monoclonal antibody is prepared from the following components in a preservation number of CCTCC: c201628. The CMG2 monoclonal antibody has the advantages of good specificity, high affinity and the like, and solves the problem of preparing CMG2 high-affinity antibodies. The anthrax toxin receptor 2 monoclonal antibody can be used for developing tumor diagnosis reagents and diagnosis reagents for classifying and typing tumors, and can be used for developing treatment drugs or drug carriers aiming at CMG2 positive tumors.

Description

Anthrax toxin receptor 2 monoclonal antibody, preparation method and application

Technical Field

The invention relates to the field of molecular biology and biotechnology, and particularly relates to an anthrax toxin receptor 2 monoclonal antibody, and a preparation method and application thereof.

Background

Anthrax toxin receptor 2(ANTXR 2; also known as capillary ymophogenesis gene 2, CMG 2). CMG2 is a single transmembrane receptor, and the coding gene is located on chromosome four, 4q21.21, and has a molecular weight of 45 kDa. CMG2 has been shown to mediate the entry of anthrax toxin into host cells, and is one of the two receptors for anthrax toxin, and the receptor with the strongest affinity for anthrax toxin. Experiments have shown that the natural ligands of CMG2 in vivo are laminin (laminin) and type IV collagen, but the physiological function of CMG2 has not yet been elucidated.

To date, the expression and significance of CMG2 in tumor cells are not reported, and previous experiments suggest that CMG2 is closely related to the dryness characteristics of GCSCs, epithelial-mesenchymal transition (EMT) phenotype and invasive metastatic capacity; the preliminary detection result of the clinical gastric cancer specimen also indicates that the high expression of CMG2 on the gastric cancer tissue is positively correlated with the stage of TNM and negatively correlated with the survival time of the patient. Thus, CMG2 may be a novel functional marker of GCSCs, and it also suggests that CMG2 may be a novel concept and target in the diagnosis and treatment of tumors (interpretation of cancer cells based on specificity of innovation. Stem Cell Rev.200Mar9; 5 (1): 66-71).

1) CMG2 may be a sternness marker for gastric cancer stem cells

CMG2(ANTXR2) is highly expressed in the gastric cancer stem cells for the first time, and is closely related to the sternness, EMT phenotype and invasive metastatic capacity of the gastric cancer stem cells. CMG2 interacts with LRP6, which activates the Wnt signaling pathway, and CMG2 has been shown to be involved in Src-Erk pathway activation, both of which induce EMT, an important event in invasive metastatic processes. It was therefore postulated that CMG2, a functional marker of gastric cancer stem cells, might activate The Wnt pathway by interacting with LRP6 and could activate The Src-Erk pathway, inducing EMT and promoting gastric cancer invasion metastasis (The LDL receptor-related protein LRP6 mediators and chemotherapy of anti-pain toxin. cell.2006Mar 24; 124(6):1141-54), as shown in FIG. 1.

2) Silencing or down-regulating CMG2 expression can obviously improve the invasive and metastatic capacity of gastric cancer stem cells

After CMG2 is silenced, the phosphorylation of p-Src 416 (positive phosphorylation site) is reduced and the phosphorylation of p-Src527 (negative phosphorylation site) is increased, which suggests that CMG2 has a positive regulation effect on Src, therefore, the effect of CMG2 in GCSCs can regulate a classical Wnt signal pathway (Wnt/β -catenin) and a non-classical Wnt/Jun kinase pathway through LRP6, and also can activate a regulatory pathway of Src-Erk.

3) CMG2 monoclonal antibody neutralizing CMG2 is an effective tumor therapy

Experiments of silencing and knocking down CMG2 prove that the effect of reducing the occurrence of EMT and inhibiting the invasion and metastasis capacity of tumors can be achieved by inhibiting CMG 2. Among the current biotherapeutic approaches, antibody therapy is the most effective and mature approach. The monoclonal antibody which is designed and prepared by taking CMG2 as a target can achieve the effect of treating the tumor, and relevant research reports exist abroad. Amit chaudhary inhibits tumor proliferation by inhibiting pathological angiogenesis by preparing a TEM8 monoclonal antibody (another receptor for anthrax toxin) (TEM8/ANTXR1Block inhibitor in vitro angiogenesis and cations tumor Responses against multiple Cancer types. Cancer Cell 21, 212-226, February 14,2012); lorna M reports that both TEM 8and CMG2 can achieve tumor treatment by inhibiting tumor angiogenesis (Targeting the anti-tumor receptors, TEM-8and CMG-2, for anti-inflammatory therapy. Through the on-line search of antibodyyparia (https:// www.antibodypedia.com /), the antibody of CMG2 is mainly polyclonal antibody, only few monoclonal antibodies are reported and the effect is unknown, the Amitchaudhary article also mentions that the monoclonal antibody of TEM8 prepared at the beginning has poor effect and fails, and finally the antibody is obtained by adopting an antibody library technology. Therefore, no report has been made on the application of the CMG 2-targeted monoclonal antibody, and the main reason is that no successful high-affinity CMG2 monoclonal antibody having a good binding effect with native CMG2 has been prepared.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide an anthrax toxin receptor 2 monoclonal antibody, a preparation method and application, solves the problem of preparation of a CMG2 high-affinity antibody, and the anthrax toxin receptor 2 monoclonal antibody can be used for developing a tumor diagnosis reagent and a diagnosis reagent for classifying and typing tumors and can be used for developing a treatment drug or a drug carrier aiming at CMG2 positive tumors.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

the anthrax toxin receptor 2 monoclonal antibody is prepared from a monoclonal antibody with a preservation number of CCTCC (China center for type culture collection): c201628.

The hybridoma cell strain is named as 8-F3, and the culture name is as follows: hybridoma cell line 8-F3(IgG1), deposited with the China center for type cultures, address: wuhan university in Wuchang Lojia mountain, Wuhan city, preservation date: 2016, 2 months and 25 days.

The anthrax toxin receptor 2 monoclonal antibody is a monoclonal antibody specific for CMG 2.

The preparation method of the anthrax toxin receptor 2 monoclonal antibody comprises the following steps:

1) immunizing Balb/c mice with purified CMG2 recombinant extracellular domain protein as an immunogen;

2) the method comprises the steps of carrying out cell fusion on inguinal lymph node cells of a Balb/c mouse and myeloma cells SP2/0 for multiple times by a hybridoma technology, screening positive clones by an indirect ELISA (enzyme-linked immunosorbent assay) method, and cloning to screen out a hybridoma cell strain capable of stably secreting CMG2 monoclonal antibody.

Further, the Balb/c mice in the step 1 are 8 weeks old, and the pelma of the Balb/c mice are immunized twice on the first day and the 9 th day respectively by using Freund's complete adjuvant and Freund's incomplete adjuvant, wherein the antigen doses of the Freund's complete adjuvant and the Freund's incomplete adjuvant are both 100 micrograms.

Further, step 2) comprises: mixing inguinal lymph node isolated cells with myeloma cells, forming fused cells with polyethylene glycol 1450 as a fusing agent, suspending the fused cells in HAT (H-Hypoxanthine Hypoxanthine, A-Aminopterin methotrexate, T-Thymidine Thymidine) culture solution containing calf serum, and juxtaposing CO₂Culturing at 37 deg.C; screening by indirect ELISA method, screening by CMG2 recombinant extracellular region protein coating, cloning detected positive cloning well by limiting dilution method, and placing in CO₂The cells were cultured at 37 ℃ until all the cells were positive in the culture medium.

A screening method of anthrax toxin receptor 2 monoclonal antibody which is best combined with natural anthrax toxin receptor 2 protein comprises the following steps:

1) paraffin sections were taken from 10 cases of gastric cancer tissues and were routinely dewaxed to water: immersing the paraffin sections in dimethylbenzene for 10min for three times; taking out paraffin section, and placing in 100% anhydrous ethanol for 10min twice; sequentially adding 90-70% alcohol for 10min each; taking out and placing in distilled water for 10 min;

2) slicing dried paraffin incubated at 37 ℃, and drawing circles with the paraffin;

3) antigen retrieval: allowing 3M urea to act at room temperature for 30 min; shaking with distilled water for 5min for three times; 0.01MpH7.4PBS is shaken for 5min for three times;

4) dropwise adding 3% H₂O₂Acting at room temperature for 10 min; shaking with distilled water for 5min for three times; washing with PBS for 10min twice;

5) adding 1% bovine serum albumin dropwise, reacting at 37 deg.C for 30min, pouring off, and washing;

6) dripping different CMG2 monoclonal antibodies with the same dilution and solubility, taking mouse Ig supernatant as negative control, and keeping the temperature at 4 ℃ overnight; shaking with PBS for 10min, three times, removing liquid (tissue is cut and not dried), and placing in a wet box;

7) dripping goat anti-mouse secondary antibody marked with HRP, and acting at room temperature for 30 min; shaking with PBS for 10min, three times, removing liquid (tissue is cut and not dried), and placing in a wet box;

8) dripping DAB working solution as color developing agent, and reacting at room temperature for 5-30min, or controlling color development under light mirror; after the color development is complete, flushing distilled water to stop color development;

9) dehydrating alcohol (70% -100%) at each stage for 10 min; taking out paraffin sections and putting the paraffin sections into dimethylbenzene for 10min and three times;

10) and (6) sealing the gum.

Further, hematoxylin counterstaining is necessary before dehydration in step 9).

Furthermore, the screened anthrax toxin receptor 2 monoclonal antibody which is best combined with the natural anthrax toxin receptor 2 protein.

The anthrax toxin receptor 2 monoclonal antibody is applied in preparing tumor diagnosis reagent and tumor classifying and typing diagnosis reagent, and the reagent is independent tumor marker.

Application of the anthrax toxin receptor 2 monoclonal antibody in preparing a therapeutic drug or a drug carrier for treating CMG2 positive tumors.

Further, the tumors include stomach cancer, colon cancer, rectal cancer, esophageal cancer, liver cancer, lung cancer, kidney cancer and breast cancer.

According to the preparation method of the anthrax toxin receptor 2 monoclonal antibody, the CMG2 monoclonal antibody capable of being combined with natural CMG2 is successfully prepared by fusing sensitized B lymphocytes of the separation lymph nodes and SP2/0 cells through an efficient immunization program, the CMG2 monoclonal antibody has the advantages of good specificity, high affinity and the like, and the problem of preparation of a CMG2 high-affinity antibody is solved; the anthrax toxin receptor 2 monoclonal antibody of the invention shows that the antibody has different detection effects on tumors in the histochemical detection: the CMG2 monoclonal antibody has histochemical staining conditions of the same tumor, namely, part of gastric cancer samples and part of breast cancer samples respectively show strong positive expression, positive expression and basically no expression, and the positive strength has no obvious relation with the classification and grading of the tumor; the CMG2 monoclonal antibody has the histochemical staining conditions of strong positive expression of partial tumors, general expression of partial tumors and no expression, and samples show strong positive expression in lung cancer, kidney cancer, breast cancer and esophageal cancer under the same conditions; the histochemical staining condition of the CMG2 monoclonal antibody in normal tissues is basically negative, the specificity of the CMG2 monoclonal antibody in tumor detection is shown, the anthrax toxin receptor 2 monoclonal antibody is almost free from target protein combination, and the CMG2 monoclonal antibody can be used as an anti-tumor medicament or a medicament carrier aiming at the positive tumor of cmG2, namely the CMG2 monoclonal antibody has the expression universality and specificity of the tumor. Meanwhile, the CMG2 monoclonal antibody has similar histochemical staining conditions in different tumors, which shows that the CMG2 monoclonal antibody has potential diagnosis and classification and typing diagnosis values, and is an independent tumor marker as a reagent for diagnosis and classification and typing diagnosis.

Drawings

FIG. 1 shows Gene chip analysis of high expression of ANTXR2(CMG2) in gastric cancer stem cells;

FIG. 2 shows partial positive results in 10 samples of gastric cancer stained with 8-F3 according to the present invention;

FIG. 3 is a schematic diagram of commercial antibody identification of CMG2 recombinant protein; among them, FIG. 3A is a reaction of a rabbit polyclonal antibody of Protech corporation with CMG2 recombinant protein; FIG. 3B is the reaction of rabbit polyclonal antibody from Sigma with CMG2 recombinant protein; FIG. 3C is the reaction of rabbit polyclonal antibodies from Protech corporation with CYCS recombinant protein; FIG. 3D is the reaction of rabbit polyclonal antibody from Sigma with CYCS recombinant protein;

FIG. 4 shows the specificity of two antibodies 3B11 and 8-F3 prepared by ELISA; wherein, fig. 4A is the reaction of 3B11 with TEM8 recombinant protein; FIG. 4B is a reaction of 3B11 with CMG2 recombinant protein; FIG. 4C is a reaction of CMG2 monoclonal antibody corresponding to 8-F3 with TEM8 recombinant protein; FIG. 4D is a reaction of CMG2 monoclonal antibody corresponding to 8-F3 with CMG2 recombinant protein;

FIG. 5 shows the results of comparison of 8-F3 of the present invention with Protech antibodies;

FIG. 6 is a graph of partial results from 8-F3 stained gastric and breast cancer samples of the present invention;

FIG. 7 shows partial positive results of the 8-F3 stained multiple tumor tissue chips according to the present invention;

FIG. 8 is a partial positive result of the 8-F3 stained normal tissue chips according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The preparation method of the anthrax toxin receptor 2 monoclonal antibody mainly comprises the following steps:

1) the purified CMG2 recombinant extracellular domain protein is used as immunogen to immunize Balb/c mice,

comprises the following steps: respectively using Freund complete adjuvant and Freund incomplete adjuvant to immunize soles of 8-week-old Balb/c mice twice on the first day and the 9 th day, wherein the dosages of the Freund complete adjuvant and the Freund incomplete adjuvant are 100 micrograms;

2) the inguinal lymph node cell of a mouse and a myeloma cell SP2/0 are subjected to cell fusion for multiple times by a hybridoma technology, an indirect ELISA method is utilized to screen positive clones, hybridoma cell strains capable of stably secreting CMG2 monoclonal antibodies are screened by cloning,

comprises the following steps: isolation of inguinal lymph nodeMixing the cells with myeloma cells, forming fused cells with polyethylene glycol 1450 as a fusing agent, suspending the fused cells in HAT culture solution containing calf serum, and juxtaposing CO₂Culturing at 37 deg.C; screening by indirect ELISA method, screening by CMG2 recombinant extracellular region protein coating, cloning detected positive cloning well by limiting dilution method, and placing in CO₂The cells were cultured at 37 ℃ until all the cells were positive in the culture medium.

EXAMPLE 1 preparation of CMG2 monoclonal antibody

1) Immunogen preparation

CMG2 protein (amino acid sequence is SEQ ID NO: 2) expressed by prokaryotic escherichia coli is used as immunogen. The specific method comprises the following steps: synthesizing a DNA sequence of the CMG2 extracellular region, introducing Nde I and Hind III (the coding sequence is SEQ ID NO: 1) enzyme cutting sites, connecting the synthesized DNA sequence of the CMG2 extracellular region with a Pet22b prokaryotic expression vector after double enzyme cutting of Nde I and Hind III, verifying whether the DNA sequence is successfully connected with a DH5a escherichia coli amplification plasmid and the double enzyme cutting verification of the amplification plasmid is successful; and transforming the successfully connected plasmid into BL21(DE3) for induced expression, purifying the corresponding recombinant protein by using a Ni column due to the fact that the expressed protein has a pre-designed His tag, and obtaining the CMG2 prokaryotic recombinant protein as an immunogen.

2) Immunogen immune Balb/c mice

The CMG2 prokaryotic recombinant protein was removed from the refrigerator, the protein concentration was determined by Lowry method (Folin-phenol reagent method), and it was diluted to 1.0mg/ml with 10mmol/L PBS (phosphate buffered saline) having a pH of 7.4. 5-10 female Balb/c mice, 8 weeks old, weighing 25g were selected. The antigen emulsification adopts an antigen emulsifier for emulsification. In the first immunization, the immunogen protein is emulsified and mixed with equal volume of Freund's complete adjuvant, and each mouse is subjected to four-limb plantar immunization according to the dose of 100 mu g of immunogen. Female Balb/c mice are immunized for the second time on day 9, Freund's incomplete adjuvant is used as the adjuvant, the antigen amount, the injection volume and the way are unchanged, and the titer is measured by an indirect ELISA method on day 21. The 5 mice with the best immune effect were selected for cell fusion.

3) Determination of serum titer of immunized Balb/c mice

Taking blood from the tail vein of the mouse 12 days after the second immunization to detect the titer of the serum antibody; with 0.1M NaCO₃(PH9.6) coating solution CMG2 prokaryotic recombinant protein is diluted to the optimal working concentration of 3-5 mug/ml, 100 mul of protein solution is added into each hole, the preservative film coated plate is kept overnight at 4 ℃, the plate is washed for 5 times by a plate washing machine, and finally the reaction plate is inverted on absorbent paper, so that the washing solution in the holes is controlled to be dry. Blood sampling and serum dilution: pinching mouse tail, disinfecting with 75% alcohol, cutting a gap in tail vein with scissors, collecting blood 20 μ l, centrifuging at 2000rpm for 30min, collecting supernatant 1 μ l, adding 99 μ l antibody diluent, mixing, diluting at a ratio of 1:100 to 1:3200, adding diluted serum to be detected 100 μ l per well, simultaneously diluting mouse serum 1:100 before immunization to serve as negative control, and using antibody diluent as blank control. Incubating for 1 hour at 37 ℃, and repeatedly washing for more than 5 times by using a plate washing machine; horse radish peroxidase goat anti-mouse IgG was diluted to 1:3000 with 0.1% PBST (phosphate buffered saline pH7.4 containing 0.05% Tween-20), added to each well in 100. mu.l, incubated at 37 ℃ for 1 hour, and washed 5 times; adding 100 mul/well of TMB (3,3',5,5' -tetramethylbenzidine) color development solution, keeping the room temperature in the dark for 1-3 minutes, adding 50 mul of stop solution into each well for observing results, stopping the color development of yellow after the TMB color development, recording the reading of 450nm by using an enzyme linked immunosorbent assay detector, and measuring that the OD value of each well is more than 2.1 times of the OD value of the negative control after the blank control wells are zeroed, thus obtaining the positive control. The serum titer reaches 1:3200, and the fusion protein can be used for cell fusion.

4) Preparation of antigen-sensitized B cells and SP2/0 cell suspension

Taking 5 Balb/c mice with the best immune effect, removing eyeballs of the mice, bleeding and killing the mice, collecting serum used as a positive control of ELISA after eye blood centrifugation, removing skin by aseptic operation, placing the mice on an aseptic operation table, separating lymph nodes with swollen limbs, placing the lymph nodes into a glass dish containing 10ml of incomplete culture medium, washing, carefully peeling peripheral connective tissues and adipose tissues, replacing the glass dish, fishing out the lymph nodes, placing the lymph nodes into a 200-mesh stainless steel net, grinding by using an inner core of an injector, washing by using the incomplete culture medium occasionally, enabling sensitized lymphocytes to penetrate through meshes to enter a solution, transferring the lymphocytes into a 10ml glass centrifuge tube, horizontally centrifuging at 1500rpm for 10min, and removing supernatant. By the same method, without usingWashing the cells 1 time with 10ml of whole culture medium, centrifuging to collect the precipitated cells, resuspending the cells in 10ml of incomplete culture medium and mixing them uniformly, the cell count is about 1 × 10⁸And (4) cells.

Taking out SP2/0 cell from liquid nitrogen, rapidly placing into 37 deg.C water bath, shaking continuously until cell solution is completely dissolved, transferring cell into 10ml centrifuge tube, horizontally centrifuging at 1500rpm for 10min, discarding supernatant, resuspending 10ml complete culture solution for precipitation, transferring cell suspension into 50ml culture flask, placing into 37 deg.C 5% CO₂Culturing in incubator, screening cells with 8-AG selective culture medium for one week after the cells grow well, transferring 1 bottle of cells to 4 bottles 2 days before fusion, blowing SP2/0 cells from the tube wall by elbow dropper, collecting in centrifuge tube, centrifuging, collecting supernatant, washing precipitate with incomplete culture medium, re-suspending with 10ml incomplete culture medium, counting cells, 5 × 10⁷。

5) Preparation of feeder cells

Taking an unimmunized Balb/c mouse, picking eyeballs, exsanguinating and killing, soaking and sterilizing with 70% ethanol for 5-10min, cutting the skin of the mouse, lifting the peritoneum with forceps, cutting a small opening with scissors, sucking ice-cold incomplete culture medium by an elbow dropper, flushing the abdominal cavity, and sucking the washing liquid into a 50ml centrifuge tube. In the same way, the abdominal cavity was washed 3 times with incomplete medium, the washings were collected, centrifuged horizontally at 1000rpm for 10min at room temperature, the supernatant was removed, and 10ml of incomplete medium was used to resuspend the cells and counted.

6) Myeloma cell and sensitized lymphocyte fusion

Before fusion, PEG 1450 (polyethylene glycol 1450) from sigma company of America is placed in an incubator at 37 ℃ for pre-heating, and 1 × 10 is sucked⁷A suspension of myeloma cells and 1 × 10⁸Adding sensitized lymphocyte suspension (cell number 1: 10) into a 50ml sterile centrifuge tube, adding 30ml RPMI-1640(Roswell Park mechanical Institute 1640) culture medium, mixing, centrifuging at 1500rpm for 10min, discarding supernatant, flicking tube bottom to make cell mass loose into paste, centrifuging tube at 37 deg.C in water bath, sucking 0.8ml of pre-warmed 50% PEG 1450 solution with dropperAdding the solution into cells slowly along the tube wall at a position about 2cm away from the bottom of the tube, rotating the centrifuge tube while adding the solution, adding the solution for about 1min, standing for 90 s, dropwise adding 30ml of RPMI-1640 culture medium pre-warmed at 37 ℃ to terminate fusion, adding the solution within 3min, slowing down and then speeding up, moving gently, standing the centrifuge tube in a 37 ℃ incubator for 5min, taking out the centrifuge tube, centrifuging at 1500rpm for 5min, discarding the supernatant, adding 10ml of HAT culture medium to resuspend the cells, blowing gently, mixing uniformly, inoculating the fused cells to 96-well cell culture plates paved with feeder cells, inoculating SP2/0 cells into each culture plate according to 100 mul/well with 6 holes reserved for each culture plate, serving as a negative control of HAT selection, placing the culture plates at 37 ℃ and 5% CO₂Culturing in an incubator.

The growth condition of the cells can be observed under an inverted microscope on the 4 th to 5 th days after fusion, and 100 mul HAT culture medium is supplemented, the hybridoma titer can be measured by an indirect ELISA method on the 10 th to 12 th days, and the HT culture medium is changed on the 14 th to 15 th days.

7) Screening of specific hybridoma cells

Detecting culture supernatant by indirect ELISA method when cells grow to 1/4-1/2 well bottoms of full culture wells 12-15 days after fusion, and screening positive clones; coating an enzyme label plate (0.3 mu g/hole) with CMG2 protein, coating overnight at 4 ℃, washing once by using a front plate washing machine, patting up liquid, adding 50 mu l of cell culture supernatant and 50 mu l of 0.2% PBST, selecting immune serum of a mouse as a positive control, selecting SP2/0 culture supernatant as a negative control, and incubating for 1h at 37 ℃ by using a washing solution as a blank control; washing the enzyme label plate: add 100. mu.l 1: 60000.1% PBST diluted HRP labeled goat anti mouse Ig antibody, 37 degrees C were incubated for 45 minutes; washing, drying, adding 100 μ l/well of TMB solution, reacting at room temperature in dark for 2-3 min, adding 50 μ l/well of stop solution to stop reaction, and detecting absorbance value of 450nm with microplate reader.

The results were: using CMG2 as an antigen, sequentially immunizing 20 Balb/c mice, fusing for 2 times, sequentially and jointly screening to obtain a CMG2 antibody 16 strain with high affinity, cloning for 4 times and repeatedly screening by ELISA to obtain a hybridoma cell strain secreting CMG2 monoclonal antibody, freezing and storing the hybridoma cells for several times, carrying out in-vitro subculture for more than 3 months, stably secreting CMG2 monoclonal antibody, and finally freezing and storing in a liquid nitrogen tank.

8) Preparation of CMG2 monoclonal antibody ascites

After screening out positive clone, immediately adopting a limiting dilution method to carry out cloning culture on the positive hybridoma cell, preparing feeder cells, using 10ml of incomplete culture medium to carry out heavy suspension, collecting the positive clone cell and counting, using the incomplete culture medium to dilute the positive clone cell to 100/20 ml, taking a 96-hole cell culture plate with the feeder cells added in advance, adding 200 mu l of cell suspension, transferring the remaining positive clone cell to a 24-hole plate to carry out expanding culture, collecting the cell liquid nitrogen for freezing, and simultaneously carrying out 5% CO freezing preservation on the culture plate at 37 DEG C₂Culturing in an incubator, observing the growth condition of the cells under a microscope after 3 days, and gradually culturing the cells in an enlarged way. Injecting sterilized paraffin oil into abdominal cavity of 8 week old mouse or multiparous mouse, and injecting 10 days later⁸The hybridoma cells are injected into the abdominal cavity of the mouse, and ascites of the mouse is obtained in a manner of extracting or killing the cells for 10-14 days for purifying the CMG2 monoclonal antibody.

9) Identification of the CMG2 monoclonal antibody subclass

The procedure was carried out as described in the United states Sigma company Mouse Monoclonal Antibody isotope Reagents (ELISA/Ouchterlony Double Diffusion, Stock No. ISO-2 LOT 114k 4817).

(1) Coating an enzyme label plate: CMG2 protein was diluted to the optimal working concentration of 3ug/ml with coating solution, 100. mu.l antigen solution per well and 12 wells per strain, overnight at 4 ℃, washed 5 times and air dried.

(2) And (3) sealing: add 5% BSA (bovine serum albumin) blocking solution 350. mu.l per well, incubate 1-1.5 h at 37 ℃, wash 5 times, air dry.

(3) Add 100. mu.l of fresh hybridoma cell culture supernatant to each well, let stand at room temperature for 1h, shake wash for 3min, 5 times in total.

(4) Dilution with antibody at 1: IgG1, IgG2a, IgG2b, IgG3, IgM, IgA were diluted 2000.

(5) Add 100. mu.l of diluted culture supernatants to each well, add 2 wells to each supernatant, stand at room temperature for 30min, shake wash for 3min, 5 times total.

(6) Add 100. mu.l 1: the rabbit anti-sheep IgG antibody diluted 5000 was allowed to stand at room temperature for 15min and then shaken for 3min for 5 times.

(7) Adding 100 mul of substrate color developing solution into each hole, and reacting for 10min-15min at room temperature in a dark place.

(8) Mu.l of stop solution was added to each well, and the Ig subclasses were determined by observing the results.

10) Purification of CMG2 monoclonal antibody in ascites

Freshly collected ascites at 3000r/min for 15min, removal of cellular components (or solid matter formed during cryopreservation), etc.; taking clear ascites at the upper layer, adding equal amount of PH7.2 barbital buffer saline (VBS; 0.004mol/L barbital, 0.15mol/L NaCl, 0.8mmol/L Mg2+, 0.3mmol/L Ca2+) for dilution; then adding 150mg of silicon dioxide powder into each 10ml of diluted ascites, mixing uniformly, and incubating the suspension for 30 minutes at room temperature, and shaking occasionally; centrifugation at 3000g for 20 min to remove lipids and the like, thus obtaining clarified ascites fluid of the CMG2 monoclonal antibody.

(1) Configuring buffer required for CMG2 monoclonal antibody purification:

binding Buffer (solution A), 100mmol/L sodium phosphate (sodium phosphate), 100mmol/L sodium citrate (sodium citrate), pH 7.0.

Elution Buffer (solution B), 100mmol/L hydrochloric acid, pH 2.7.

Assembly Buffer (solution C) 1mol/L Tris (hydroxymethyl) aminomethane (Tris-HCl), pH 9.0.

(2) Ascites of the CMG2 monoclonal antibody was mixed with fluid a at a ratio of 1:10 and filtered through a 0.45 μm filter to be loaded.

(3) A HiTrap rProtein A HP column is selected to be connected to AKTA Explorer, and the A liquid and the B liquid are fully balanced in respective pipelines.

(4) And (3) loading the sample prepared in the step (2) from the tube A, balancing the column by using the liquid A after loading, eluting the purification column by using the liquid B, and collecting an elution peak to a collection tube in which a small amount of liquid C is added in advance.

(5) Adjusting the pH of the eluted protein to 7.0-8.0, and freezing and storing the CMG2 monoclonal antibody in separate packages.

Example 2 screening of CMG2 monoclonal antibodies that bind optimally to native CMG2 protein

An immunohistochemical technical scheme, which is to screen the optimal CMG2 monoclonal antibody capable of optimally combining with natural CMG2 protein from a group of CMG2 monoclonal antibodies.

Specifically, 10 cases of paraffin sections of gastric cancer tissues stored in the pathology department of the first subsidiary hospital of the third military medical university of the Chinese people's liberation force (purchased from the first subsidiary hospital of the third military medical university of the Chinese people's liberation force) are used for detecting the immunoreactivity of the prepared CMG2 monoclonal antibody to natural CMG2, and a CMG2 monoclonal antibody strain with the best histochemical effect under the same conditions is selected, and the method comprises the following steps:

1) taking a paraffin section of the gastric cancer tissue, and dewaxing the paraffin section to water conventionally: immersing the paraffin sections in dimethylbenzene for 10min for three times; taking out paraffin section, and placing in 100% anhydrous ethanol for 10min twice; sequentially adding 90-70% alcohol for 10min each; taking out and placing in distilled water for 10 min.

2) And (4) hatching dry paraffin sections at 37 ℃, and drawing circles by using the paraffin.

3) Antigen retrieval: allowing 3M urea to act at room temperature for 30 min; shaking with distilled water for 5min for three times; 0.01M pH7.4PBS shake wash for 5min, three times.

4) Dropwise adding 3% H₂O₂Acting at room temperature for 10 min; shaking with distilled water for 5min for three times; the PBS was shaken for 10min twice.

5) Adding 1% bovine serum albumin dropwise, reacting at 37 deg.C for 30min, pouring off, and washing-free.

6) Dripping different CMG2 monoclonal antibodies with the same dilution and solubility, taking mouse Ig supernatant as negative control, and keeping the temperature at 4 ℃ overnight; PBS was shaken for 10min, three times, the liquid was spun off (tissue cut without drying) and placed flat in a wet box.

7) Dripping goat anti-mouse secondary antibody marked with HRP, and acting at room temperature for 30 min; PBS was shaken for 10min, three times, the liquid was spun off (tissue cut without drying) and placed flat in a wet box.

8) Dripping DAB working solution as color developing agent, and reacting at room temperature for 5-30min, or controlling color development under light mirror; after the color development is completed, the distilled water is flushed to stop the color development.

9) Hematoxylin counterstain if necessary; dehydrating alcohol (70% -100%) at each stage for 10 min; taking out paraffin sections and putting the paraffin sections into dimethylbenzene for 10min, three times.

10) And (6) sealing the gum.

10 paraffin sections of gastric cancer tissues were stained with one group of prepared CMG2 monoclonal antibodies, and the CMG2 monoclonal antibody having the most significant effect in comparison with the same concentration and dilution was considered to be the CMG2 monoclonal antibody that binds the most to the native CMG2 protein.

The hybridoma cell strain of the best CMG2 monoclonal antibody screened by the invention is named as 8-F3, and the preservation number is CCTCC NO: 201628, culture name: hybridoma cell strain 8-F3(IgG1), the preservation unit is CCTCC for short, address: wuhan university in Wuhan (Wuhan university in Wuchang Lojia mountain) in Wuhan City, the preservation date is No. 2/25 in 2016.

FIG. 2 shows that, when the prepared CMG2 monoclonal antibody is diluted to the same concentration and stained with 10 paraffin sections of gastric cancer tissues, the CMG2 monoclonal antibody corresponding to 8-F3 has the best staining effect and the highest positive rate under the same conditions.

Example 3 identification of specificity of CMG2 monoclonal antibody

1) Commercial antibody validation of CMG2 recombinant protein

The effectiveness of the prepared CMG2 antigen was verified by the purchased rabbit polyclonal antibody from Protech (cat # 16723-1-AP) and the rabbit polyclonal antibody from sigma (cat # SAB 1400784). Coating CMG2 recombinant protein and irrelevant CYCS recombinant protein expressed and purified in the same way: the CMG2 recombinant protein and CYCS recombinant protein were diluted to 3. mu.g/ml with coating diluent, and 100. mu.l of each ELISA plate was added to each well and coated overnight at 4 ℃. Taking out the plate the next day, abandoning the antigen, and washing the plate. The purchased rabbit polyclonal antibody from Protech and the rabbit polyclonal antibody from sigma were made into 1: 1000,1: 2000,1: 4000,1: 8000,1: 16000 … …, adding into the corresponding CMG2 recombinant protein and CYCS recombinant protein strip wells at a volume of 100 μ l/well, and making blank and yin-yang control wells. Incubate at 37 ℃ for 1h, wash plate, add secondary antibody, 1:3000 HRP-labeled goat anti-rabbit IgG was added to each well at 100. mu.l/well and incubated at 37 ℃ for 40 min. Discarding the solution, washing the plate, patting to dry, adding 100 mu l/hole of TMB substrate solution, performing dark color development for 10min, and adding 50 mu l/hole of stop solution. The results are shown in FIGS. 3A-3D, where FIG. 3A shows that the reaction between the rabbit polyclonal antibody from Protech and the CMG2 recombinant protein is better; FIG. 3B shows that the reaction of rabbit polyclonal antibody from Sigma with CMG2 recombinant protein is better; FIG. 3C shows the reaction of rabbit polyclonal antibody from Protech with CYCS recombinant protein, resulting in no reaction; FIG. 3D shows the reaction of rabbit polyclonal antibody from Sigma with CYCS recombinant protein, resulting in no reaction. That is, both rabbit polyclonal antibodies from Protech and rabbit polyclonal antibodies from sigma recognize the CMG2 recombinant protein well, but do not react with the CYCS recombinant protein.

2) ELISA for identifying specificity of CMG2 monoclonal antibody

The TEM8 recombinant protein is another receptor protein of anthrax toxin, has similar functions and higher protein homology with CMG2, expresses and recombines the prepared TEM8 protein by the same expression mode, and identifies whether the protein can be combined with the TEM8 recombinant protein. The TEM8 recombinant protein and the CMG2 recombinant protein were diluted to 3. mu.g/ml with coating diluent, 100. mu.l of each well of the ELISA plate was added, and the plate was coated overnight at 4 ℃. Taking out the plate the next day, abandoning the antigen, and washing the plate. Monoclonal antibodies 3B11, 8-F3 were made as 1: 1000,1: 2000,1: 4000,1: 8000,1: 16000 … …, adding into corresponding wells at a volume of 100 μ l/well, and making blank and yin-yang control wells. Incubate at 37 ℃ for 1h, wash plate, add secondary antibody, 1:3000 HRP-labeled goat anti-mouse IgG was added to each well at 100. mu.l/well and incubated at 37 ℃ for 40 min. Discarding the solution, washing the plate, patting to dry, adding 100 mu l/hole of TMB substrate solution, performing dark color development for 10min, and adding 50 mu l/hole of stop solution. The results are shown in FIGS. 4A-4D, where FIG. 4A is the reaction of 3B11 with TEM8 recombinant protein and no reaction results; FIG. 4B is the reaction of 3B11 with CMG2 recombinant protein, resulting in reaction and dilution; FIG. 4C shows the reaction of CMG2 monoclonal antibody corresponding to 8-F3 with TEM8 recombinant protein, resulting in no reaction; FIG. 4D is a reaction of CMG2 monoclonal antibody corresponding to 8-F3 with CMG2 recombinant protein, resulting in reaction and dilution. Namely, the CMG2 monoclonal antibody corresponding to 8-F3 can better recognize CMG2 recombinant protein, but not recognize the same TEM8 recombinant protein with His tag, and has better specificity.

3) Immunohistochemistry comparison of specificity of the CMG2 monoclonal antibody

Selecting and purchasing CMG2 polyclonal antibody of Protech company with best evaluation effect abroad, adopting instruction conditions and optimum conditions of 8-F3 for groping, staining the same tumor tissue sections respectively, comparing staining conditions, and determining whether the specificity is consistent. The specific operation brief introduction is as follows:

(1) paraffin sections of stomach, colon, and rectal cancers were routinely dewaxed to water in two portions: immersing the paraffin sections in dimethylbenzene for 10min for three times; taking out paraffin section, and placing in 100% anhydrous ethanol for 10min twice; sequentially adding 90-70% alcohol for 10min each; taking out and placing in distilled water for 10 min.

(2) And (4) hatching dry paraffin sections at 37 ℃, and drawing circles by using the paraffin.

(3) Antigen retrieval: allowing 3M urea to act at room temperature for 30 min; shaking with distilled water for 5min for three times; 0.01M pH7.4PBS shake wash for 5min, three times.

(4) Dropwise adding 3% H₂O₂Acting at room temperature for 10 min; shaking with distilled water for 5min for three times; the PBS was shaken for 10min twice.

(5) Adding 1% bovine serum albumin dropwise, reacting at 37 deg.C for 30min, pouring off, and washing-free.

(6) Dripping 8-F3 and CMG2 polyclonal antibody of Protech company, taking corresponding species IgG as negative control, and staying overnight at 4 ℃; PBS was shaken for 10min, three times, the liquid was spun off (tissue cut without drying) and placed flat in a wet box.

(7) Dripping a corresponding secondary antibody marked with HRP, and acting for 30min at room temperature; PBS was shaken for 10min, three times, the liquid was spun off (tissue cut without drying) and placed flat in a wet box.

(8) Dripping DAB working solution as color developing agent, and reacting at room temperature for 5-30min, or controlling color development under light mirror; after the color development is completed, the distilled water is flushed to stop the color development.

(9) Hematoxylin counterstain if necessary; dehydrating alcohol (70% -100%) at each stage for 10 min; taking out paraffin sections and putting the paraffin sections into dimethylbenzene for 10min, three times.

(10) And (6) sealing the gum.

The results are shown in FIGS. 5A-5B, in which FIG. 5A shows, from left to right, the negative control of CMG2 monoclonal antibody corresponding to 8-F3 and the positive results of the staining of gastric cancer, colon cancer and rectal cancer; FIG. 5B shows, from left to right, a negative control of the Protech CMG2 polyclonal antibody and positive results of the negative control staining gastric cancer, colon cancer and rectal cancer, respectively; the results show that the CMG2 monoclonal antibody corresponding to 8-F3 of the invention has a staining effect similar to that of the CMG2 polyclonal antibody of Protech company, nearly consistent staining is performed on gastric cancer, colon cancer and rectal cancer, and the results are consistent in a staining area and a staining location, so that the CMG2 monoclonal antibody corresponding to 8-F3 of the invention can replace the CMG2 polyclonal antibody of Protech company. The CMG2 monoclonal antibody of the invention is proved to have better effect and specificity for recognizing CMG2, and is the CMG2 specific monoclonal antibody.

Example 4 tumor diagnostic applications of the CMG2 monoclonal antibody

The early research of CMG2 was mainly directed to its role in the attack of anthrax toxin, and only the preparation of monoclonal antibodies to block its receptor action was patented to reduce the damage caused by anthrax toxin to humans. The stem cell research experiments show that the expression difference of the CMG2 monoclonal antibody in the differential expression of tumor stem cells and non-tumor stem cells is very obvious, which suggests that the CMG2 monoclonal antibody may have an important function in tumors, the effect of inhibiting the tumors may be achieved by blocking or knocking down CMG2 molecules, and the CMG2 monoclonal antibody may be an important target point for anti-tumor therapy. The CMG2 monoclonal antibody is found to have the expression universality and specificity of tumors in the histochemical staining of a series of tumor samples through the 8-F3. The main application studies and results are as follows:

1) histochemical staining of CMG2 monoclonal antibody in homogeneous tumors

The 8-F3 of the invention is used for carrying out histochemical staining on 200 paraffin sections of gastric cancer and breast cancer tissues preserved in the pathology department of the first subsidiary hospital of the third military medical university of the national liberation force (purchased from the first subsidiary hospital of the third military medical university of the national liberation force) by adopting the same detection conditions, and analyzing the expression condition of the CMG2 monoclonal antibody in the same tumor. 8-F3 was diluted to optimal concentrations and stained with 200 samples of gastric cancer and breast cancer, respectively, and the results are shown in FIGS. 6A-6F, wherein FIGS. 6A-6C show that the CMG2 monoclonal antibody corresponding to 8-F3 has different expression patterns in gastric cancer, and FIGS. 6D-6F show that the CMG2 monoclonal antibody corresponding to 8-F3 has different expression patterns in breast cancer. Fig. 6A and 6D show that some samples of gastric cancer and breast cancer show strong positive expression under the same conditions, fig. 6B and 6E show that some samples of gastric cancer and breast cancer show positive expression, and fig. 6C and 6F show that some samples of gastric cancer and breast cancer show no substantial expression. The result also shows that the positive strength has no obvious relation with the classification and grading of the tumor, and the CMG2 monoclonal antibody is possibly an independent tumor marker.

2) Histochemical staining of the CMG2 monoclonal antibody in other tumors

The purchased tumor tissue chip (multi-organ, OD-CT-Com04-001) of Chi-super company contains 7 kinds of multi-organ cancers, including 5 cases of esophageal cancer, 6 cases of gastric cancer, 5 cases of colon cancer, 6 cases of liver cancer, 6 cases of lung cancer, 6 cases of breast cancer, 6 cases of kidney cancer and 7 cases of various paracarcinoma. The chip was stained with 8-F3 for histochemical expression in other tumors. 8-F3 is diluted to an optimal concentration, a tumor tissue chip is stained, the 8-F3 grouping result shows that different tumors have different degrees of expression, part of tumors have strong positive expression, part of tumors have general expression, and the cases of no expression exist at the same time, but under the same conditions, samples show strong positive expression in lung cancer, kidney cancer, breast cancer and esophageal cancer, the result is shown in figure 7, and the result shows that the CMG2 monoclonal antibody can be used for diagnosis of tumors and diagnosis of classification and typing of tumors.

3) Histochemical staining of the CMG2 monoclonal antibody in Normal tissue

The chip was stained with 8-F3 in a organized manner to see the expression of the chip in 23 tissues including the normal tissues of a plurality of persons, including the brain, cerebellum, brainstem, tongue, thyroid, esophagus, stomach, intestine, liver, pancreas, lung, trachea, appendix, skin, muscle, striated muscle, left heart, right heart, aorta, testis, bladder, prostate, and the like, using a commercially available Normal tissue chip (Multi-organ, OD-NH-Com01-001) of Sorbon. 8-F3 was diluted to the optimal concentration and stained on a normal tissue chip, the result is shown in FIG. 8, which shows that the histochemical staining in normal tissue is basically negative, and the specificity of CMG2 monoclonal antibody in tumor detection is shown. In the 8-F3 histochemical result, except that part of tissues are easy to be non-specifically colored, the tissues of other normal people are almost not colored, which indicates that 8-F3 has combination on the expression of CMG2 under pathological conditions, has better tumor specificity and is a good target treatment drug or drug carrier aiming at the positive tumor of CMG 2.

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. An anthrax toxin receptor 2 monoclonal antibody is characterized in that the preservation number is CCTCC: c201628.

2. The anthrax toxin receptor 2 monoclonal antibody of claim 1, wherein the anthrax toxin receptor 2 monoclonal antibody is a monoclonal antibody specific for CMG 2.

3. The anthrax toxin receptor 2 monoclonal antibody of claim 1 or 2 for use in the preparation of a diagnostic reagent for tumor diagnosis and a diagnostic reagent for classification and typing of tumors.

4. Use of the anthrax toxin receptor 2 monoclonal antibody of claim 1 or 2 in the preparation of a therapeutic agent or a pharmaceutical carrier for the treatment of CMG2 positive tumors.

5. Use according to claim 4, characterized in that: the tumor includes gastric cancer, colon cancer, rectal cancer, esophageal cancer, liver cancer, lung cancer, kidney cancer and breast cancer.