CN117821400A - TFF1 monoclonal antibody for inhibiting breast cancer bone metastasis and preparation method and application thereof - Google Patents
TFF1 monoclonal antibody for inhibiting breast cancer bone metastasis and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a TFF1 monoclonal antibody for inhibiting breast cancer bone metastasis. The monoclonal antibody has a preservation number of CCTCC: hybridoma cell line preparation of C2023397. The TFF1 monoclonal antibody has the advantages of good specificity, high affinity and the like. The TFF1 monoclonal antibody can be used for inhibiting breast cancer bone metastasis and researching molecular mechanism of inhibiting breast cancer bone metastasis by using the TFF 1.
Description
Technical Field
The invention relates to the fields of molecular and molecular biology and biotechnology, in particular to a TFF1 monoclonal antibody for inhibiting breast cancer bone metastasis, and a preparation method and application thereof.
Background
The clover factor 1 (Trefoil Factor Family, TFF1, also called D21S21, HPS2, pS2, etc.) coding gene is located on chromosome 21 (chr 21:42,352,293-42,356,546), and is composed of 84 amino acids with a molecular weight of 9.15kDa. TFF1 was first isolated from the human Luminal breast cancer cell line MCF-7 and identified as an estrogen-regulated gene. Under normal physiological state, TFF1 exists mainly in digestive system tissues such as stomach, intestine and the like, and plays roles in mucous membrane protection, tissue injury repair and immune response of gastrointestinal parts.
Breast cancer is the most frequently occurring malignancy worldwide, and the luminel type is the predominant molecular typing (-60%). Although overall prognosis of luminel breast cancer is superior to other molecular types, it has the characteristics of insensitivity to chemotherapy and relatively long survival time, high accumulation distant metastasis rate, and especially easy occurrence of bone metastasis (about 75%), and bone-related events caused by bone metastasis and secondary metastasis to the lung and brain seriously threaten the life and quality of life of patients, which is an important problem to be solved in clinical urgent need.
More and more clinical studies in recent years show that TFF1 is closely related to breast cancer bone metastasis caused by chemotherapy or endocrine therapy drug resistance. There is no commercially available monoclonal antibody directed against TFF1, which makes it difficult to formulate a therapeutic strategy for inhibiting the occurrence of breast cancer bone metastases by inhibiting TFF1, a potential therapeutic target for tumors.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a TFF1 monoclonal antibody, a preparation method and application thereof, solves the problem of preparing a TFF1 high-affinity antibody, and can be used for inhibiting breast cancer bone metastasis and researching a molecular mechanism of inhibiting breast cancer bone metastasis by using the TFF1 monoclonal antibody. Meanwhile, the kit can also be used for detecting and sorting flow cells of breast cancer bone metastasis and can be used as a diagnosis index of immunohistochemical of breast cancer bone metastasis.
In order to achieve the above object, the present invention adopts the following technical scheme:
the invention provides a hybridoma cell strain for preparing a TFF1 monoclonal antibody, which has a preservation number of CCTCCNO: C2023397. The hybridoma cell line was designated as Hu-T1, culture designation: hybridoma cell line Hu-T1 (Hybridoma cell line Hu-T1), deposited by China center for type culture Collection, address: the university of Wuhan mountain and Wuhan of Wuhan city, wuchang Lopa, preservation date: 2023, 12, 21.
The invention also provides a TFF1 monoclonal antibody secreted and prepared by the hybridoma cell strain. The TFF1 monoclonal antibody is 7G11. The TFF1 monoclonal antibody is a TFF1 specific monoclonal antibody.
The invention also provides a preparation method of the TFF1 monoclonal antibody, which mainly comprises the following steps:
1) Designing and preparing a TFF1 immunogen polypeptide;
2) Immunizing a Balb/c mouse with the prepared immunogenic polypeptide;
3) The inguinal lymph node cells of the Balb/c mice in the step 2) and myeloma cells SP2/0 are subjected to multiple cell fusion by using a hybridoma technology, positive clones are screened by using an indirect ELISA method, and hybridoma cell strains capable of stably secreting the TFF1 monoclonal antibodies are screened in a cloning mode;
4) Preparing TFF1 monoclonal antibody ascites;
5) Purifying the TFF1 monoclonal antibody;
6) Identification of the TFF1 monoclonal antibody subclass;
7) Identification of the specificity of the TFF1 monoclonal antibody.
The step 1) further comprises: the immunogenic polypeptide sequence site is designed in a region with better surface accessibility, flexibility, hydrophilicity and the like. Preferably, the designed immunogenic polypeptide is TFF1-epitope69, the corresponding polypeptide sequence site being: 69-82 as shown in SEQ ID NO. 1 (FYPNTIDVPPEEEC).
The step 2) further comprises: the Balb/c mice were 8 week old, female, normothermic mice.
The step 3) further comprises: performing primary immunization, secondary immunization, tertiary immunization and boosting immunization on the Balb/c mouse, wherein the primary immunization is to emulsify and mix the TFF1 immunogen polypeptide prepared in the step 1) with an equal volume of Freund's complete adjuvant to prepare immunogens, the secondary immunization and the tertiary immunization are to emulsify and mix the TFF1 immunogen polypeptide prepared in the step 1) with an equal volume of Freund's incomplete adjuvant to prepare immunogens, the boosting immunization is to emulsify and mix the TFF1 immunogen polypeptide prepared in the step 1) to prepare immunogens, and the dosage and the administration route of each immunogen are unchanged.
The step 3) further comprises: mixing inguinal lymph node separated cells with myeloma cells, using polyethylene glycol 1450 as fusion agent to form fusion cells, suspending the fusion cells in HAT culture solution containing calf serum, and juxtaposing 5% CO 2 The cells were cultured in a cell incubator at 37 ℃. Screening by the indirect ELISA method, wherein during screening, eukaryotic expression TFF1 is used for coating and screening, and the cell cloning is carried out on the cell growth holes of the detected positive clones by adopting a limiting dilution method until the culture solution of all the cell growth holes is positive.
Preferably, in the step 6), the TFF1 monoclonal antibody subclass is an Ig subclass.
The method of the invention comprises detecting the strength of the TFF1 monoclonal antibody in inhibiting the transfer of breast cancer cells to bone cells through a cell migration experiment, and screening out the TFF1 monoclonal antibody inhibiting the bone transfer of breast cancer, and comprises the following steps:
(1) Taking human osteoblast hFOB 1.19 in logarithmic growth phase according to 2×10 4 Inoculating into 24-well Transwell cell culture plate, culturing in DMEM/F12 culture solution containing 100U/mL penicillin, 100 μg/mL streptomycin, 0.3mg/mL G418 and 10% foetal calf serum, and placing at 37deg.C and 5% CO 2 Culturing for 24h.
(2) The TFF1 monoclonal antibody or negative control equivalent negative control lgG was added to the adherent hFOB 1.19 cells, respectively, and the culture was continued for 12h.
(3) Taking human breast cancer resistant cell MCF-7/DOC (CN 111363721A) in logarithmic growth phase according to 2×10 4 Inoculating/plating into a Transwell chamber, culturing in DMEM/F12 culture solution containing 100U/mL penicillin, 100 μg/mL streptomycin, 0.3mg/mL G418 and 10% foetal calf serum, and placing at 37deg.C and 5% CO 2 And (3) respectively adding the TFF1 monoclonal antibody or negative control equivalent negative control lgG into the cell wall for 8 hours, and continuously culturing for 12 hours.
(4) Cell fixation to the outside of the Transwell chamber. The Transwell chamber was removed, the culture medium was removed, and the cells in the chamber were gently rubbed with a cotton swab or cotton soaked with PBS. 600 μl of 4% paraformaldehyde fixative was added to a clean well of a 24-well plate and the chamber was placed in the fixation for 30 minutes. The fixative was discarded and the cells were washed 1 time inside and outside the chamber with PBS.
(5) Staining and observation of the migrated cells. 600uL of crystal violet staining solution was added to a clean well of a 24-well plate and the chamber was placed for 10 minutes. The cells were removed and washed 3 times with PBS. After proper air drying, observing qualitative research under a microscope; the quantitative study was performed by taking 3-5 fields of view, taking pictures and averaging with ImageJ counts.
The application of the TFF1 monoclonal antibody in preparing a medicament for treating the transfer of breast cancer cells to bone cells can inhibit the transfer of breast cancer cells to bone cells caused by TFF1 in a mouse body.
The beneficial effects of the invention are as follows:
the specific mouse anti-human monoclonal antibody aiming at TFF1 is designed and prepared by taking breast cancer bone metastasis cells as model cells. The TFF1 monoclonal antibody 7G11 for inhibiting the occurrence of breast cancer bone metastasis is successfully constructed, a treatment strategy taking the TFF1 as a target point is provided for breast cancer bone metastasis treatment, and the TFF1 monoclonal antibody is used for researching the molecular mechanism of inhibiting breast cancer bone metastasis.
Drawings
FIGS. 1A-1B are schematic diagrams of in vitro inhibition of breast cancer cells by TFF1 monoclonal antibody 7G11. FIG. 1A shows a TFF1 monoclonal antibody 7G11 treated group and FIG. 1B shows a negative control group.
Fig. 1C is a statistical plot of fig. 1A and 1B, showing that the breast cancer cell migration ability of the TFF1 monoclonal antibody 7G11 treated group was significantly inhibited.
FIGS. 2A and 2B are graphs showing the results of in vivo inhibition of bone metastasis of breast cancer by TFF1 monoclonal antibody 7G11.
Detailed Description
The present invention will be described in further detail with reference to the following examples and drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Preservation information of the hybridoma cell lines: the preservation unit is China center for type culture collection, the address is university of Wuhan in Wuhan, han and Wuchang, lujia, hubei province, the preservation date is 2023, 12, 21, and the preservation number is CCTCC NO: c2023397, culture name and noted identification features: anti-human TFF1 hybridoma cell Hu-T1.
The preparation method of the TFF1 monoclonal antibody comprises the following steps: TFF1 polypeptide is designed as an immunogen in a region with better surface accessibility, flexibility, hydrophilicity and the like of the TFF1, a hybridoma antibody cell strain is prepared by a cell fusion technology, a corresponding TFF1 monoclonal antibody is purified, the strength of the TFF1 monoclonal antibody in inhibiting the transfer effect of breast cancer cells to the bone cell side is detected by a cell migration experiment, and the TFF1 monoclonal antibody inhibiting the bone transfer of the breast cancer is screened.
The reagents of this example were commercially available products unless otherwise specified.
Example 1: preparation of TFF1 monoclonal antibody
(1) Design and preparation of TFF1 immunogenic polypeptides
The immunogenic polypeptide sequence site is designed in a region with better surface accessibility, flexibility, hydrophilicity and the like. Namely, the designed immunogen polypeptide TFF1-epitope69, the corresponding polypeptide sequence site is: 69-82 FYPNTIDEVPPEEEC (SEQ ID NO: 1).
TABLE 1 amino acid sequence of TFF1 immunogenic peptides
| Immunogenic polypeptides | Polypeptide site | Sequence (N '-C') |
| TFF1-epitope69 | 69-82 | FYPNTIDVPPEEEC |
The above 3 immunogenic polypeptides were synthesized by solid-phase polypeptide synthesis. The specific method comprises the following steps: coupling protective amino, namely Fmoc (Aba Ding Shenghua technology Co., F483120) as a protective group of amino acid alpha-amino, coupling a first protective amino group with an external solid phase carrier MBHA resin (Aba Ding Shenghua technology Co., M466476) by a covalent bond, taking the N end of the amino acid as a synthesis starting point, reacting with excessive activated second amino acid by removing the amino protective group, splicing long peptide chains according to the sequence shown in the table 1, repeating the operation, finally splitting the peptide chains from the resin, and separating and purifying to obtain the target polypeptide.
(2) Preparation of immunogen Balb/c mice were immunized. The protein content of the TFF1-epi 69 immunogen polypeptide was measured by the Lowry method (Folin-phenol reagent method) after dissolution, and diluted to 1.0mg/ml with 10mmol/L PBS (phosphate buffer saline) at pH 7.4. Female warp-born Balb/c mice at 8 weeks of age. The Balb/c mice are subjected to primary immunization, secondary immunization and tertiary immunization, wherein the primary immunization is to emulsify and mix the TFF1 immunogenic peptide prepared in the step (1) with an equal volume of Freund's complete adjuvant (P2036, inc.), the secondary immunization and the tertiary immunization are to emulsify and mix the TFF1 immunogenic peptide prepared in the step (1) with an equal volume of Freund's incomplete adjuvant (P2031, inc.), respectively, to prepare immunogens, and the boosting immunization is to emulsify and mix the TFF1 immunogenic peptide prepared in the step (1) to prepare immunogens, wherein the dosage and the administration route of each immunogen are unchanged.
(3) Mixing inguinal lymph node cells of Balb/c mouse with myeloma cells by hybridoma technique, taking polyethylene glycol 1450 (Proteubtech, PR 40010) as fusion agent to form fusion cells, suspending the fusion cells in HAT (Aba Ding Shenghua technology Co., H475856) culture solution containing calf serum, and juxtaposing 5% CO 2 The cells were cultured in a cell incubator at 37 ℃. Screening by the indirect ELISA method, wherein during screening, eukaryotic expression TFF1 is used for coating and screening, and the cell cloning is carried out on the cell growth holes of the detected positive clones by adopting a limiting dilution method until the culture solution of all the cell growth holes is positive.
(4) And preparing TFF1 monoclonal antibody ascites. After positive clones are selected, the positive hybridoma cells are subjected to cloning culture by adopting a limiting dilution method immediately, feeder cells are prepared, 10ml of incomplete culture medium is used for resuspension, the positive clone cells are collected and counted, the incomplete culture medium is used for diluting the positive clone cells to 100/20 ml, a 96-well cell culture plate with the feeder cells added in advance is taken, 200 mu l of cell suspension is added, the rest positive clone cells are transferred into a 24-well plate for expansion culture, the collected cells are frozen in liquid nitrogen, and meanwhile, the culture plate is subjected to expansion culture at 37 ℃ and 5% CO 2 Culturing in an incubator, observing the growth condition of cells under a microscope after the 3 rd day, and gradually expanding and culturing the cells. Mice of 8 weeks of age or mice produced were injected intraperitoneally with sterilized paraffin oil (Aba Ding Shenghua technologies Co., P104801) and 10 days later 8 The hybridoma cells were injected into the abdominal cavity of the mice, and the ascites of the mice was obtained by aspiration or cell killing at 2 weeks for purification of the antibodies.
(5) Purifying the TFF1 monoclonal antibody. Freshly collected ascites fluid, 3000 rpm/min for 15 minutes, removal of cellular components (or solids formed during cryopreservation), and the like; taking clear ascites of upper layer, adding equal amount of pH 7.2 barbital buffer saline (VBS; 0.004mol/L barbital, 0.15mol/L NaCl,0.8mmol/L Mg) 2+ ，0.3mmol/L Ca 2+ ) Diluting; then diluted at every 10mlAdding 150mg of silicon dioxide powder into ascites, uniformly mixing, incubating the suspension for 30 minutes at room temperature, and shaking from time to time; after centrifugation at 3000g for 20 minutes, lipid and the like were removed by this method, clear ascites was obtained, and antibody purification was performed using HiTraprProteinAHP column from GE healthcare.
1) Configuration of buffer required for antibody purification:
binding Buffer (A) 100mmol/L sodium phosphate (sodium phosphate), 100mmol/L sodium citrate (sodium citrate), pH 7.0.
Solution B100 mmol/L sodium phosphate, 100mmol/L sodium citrate, and pH 3.0.
Assemble Buffer (solution C) 1mol/L Tris (hydroxymethyl) aminomethane (Tris-HCl), pH9.0.
2) Mixing the monoclonal antibody ascites with the A solution according to the proportion of 1:10, and filtering with a 0.45 μm filter membrane to wait for loading.
3) HiTraprProteinAHP column is selected to be connected with AKTAExplorer and A, B liquid to fully balance the pipelines.
4) Loading the sample prepared in the step 2) from a pipe A, balancing the column by using the liquid A after loading, eluting the purification column by using the liquid B, and collecting eluting peaks into a collecting pipe which is added with a small amount of liquid C in advance.
5) Adjusting pH of the eluted protein to 7.0-8.0, and freeze-preserving the antibody.
(6) A TFF1 monoclonal antibody for use in screening for inhibition of breast cancer bone metastasis. Detecting the strength of the TFF1 monoclonal antibody in inhibiting the transfer of breast cancer cells to bone cells through a cell migration experiment, and screening the TFF1 monoclonal antibody for inhibiting the bone transfer of breast cancer comprises the following steps:
1) Taking human osteoblast hFOB 1.19 in logarithmic growth phase according to 2×10 4 Inoculating into 24-well Transwell cell culture plate, culturing in DMEM/F12 culture solution containing 100U/mL penicillin, 100 μg/mL streptomycin, 0.3mg/mL G418 and 10% foetal calf serum, and placing at 37deg.C and 5% CO 2 Culturing for 24h.
2) The TFF1 monoclonal antibody or negative control equivalent negative control lgG was added to the adherent hFOB 1.19 cells, respectively, and the culture was continued for 12h.
3) Taking human breast cancer resistant cell MCF-7/DOC (CN 111363721A) in logarithmic growth phase according to 2×10 4 Inoculating/plating into a Transwell chamber, culturing in DMEM/F12 culture solution containing 100U/mL penicillin, 100 μg/mL streptomycin, 0.3mg/mL G418 and 10% foetal calf serum, and placing at 37deg.C and 5% CO 2 And (3) respectively adding the TFF1 monoclonal antibody or negative control equivalent negative control lgG into the cell wall for 8 hours, and continuously culturing for 12 hours.
4) Cell fixation to the outside of the Transwell chamber. The Transwell chamber was removed, the culture medium was removed, and the cells in the chamber were gently rubbed with a cotton swab or cotton soaked with PBS. 600 μl of 4% paraformaldehyde fixative was added to a clean well of a 24-well plate and the chamber was placed in the fixation for 30 minutes. The fixative was discarded and the cells were washed 1 time inside and outside the chamber with PBS.
5) Staining and observation of the migrated cells. 600uL of crystal violet staining solution was added to a clean well of a 24-well plate and the chamber was placed for 10 minutes. The cells were removed and washed 3 times with PBS. After proper air drying, observing qualitative research under a microscope; the quantitative study was performed by taking 3-5 fields of view, taking pictures and averaging with ImageJ counts. The TFF1 monoclonal antibody which inhibits the bone metastasis of the breast cancer most significantly is optimal. The 7G11 antibody is the optimal antibody for screening.
(7) Identification of the 7G11 subclass of TFF1 monoclonal antibody. The procedure was performed as described using the United states Sigma company Mouse Monoclonal Antibody Isotyping Reagents (ELISA/Ouchterlony Double Diffusion, stockNo. ISO-2LOT 114k 4817).
1) And (3) coating an ELISA plate: TFF1 was released to an optimal working concentration of 5ug/ml with coating solution, 100. Mu.l 7G11 antigen solution was added to each well, 12 wells were added to each strain, washed 5 times overnight at 4℃and air dried.
2) Closing: each well was incubated with 350. Mu.l of blocking solution at 37℃for 1-1.5 hours, washed 5 times and air dried.
3) Mu.l of fresh hybridoma cell culture supernatant or diluted mab was added to each well, and the mixture was allowed to stand at room temperature (20 ℃ -25 ℃) for 1 hour, and was shaken for 3 minutes 5 times.
4) Dilution with antibody at 1: igG1, igG2a, igG2b, igG3, igM, igA were diluted 2000.
5) 100 μl of diluted antibodies were added to each well, 2 wells were added to each antibody, and the mixture was allowed to stand at room temperature for 30 minutes and then shaken for 3 minutes 5 times.
6) Diluted rabbit anti-sheep IgG antibody was added 100 μl per well (1: 1000 Standing for 15 minutes at room temperature, shaking for 3 minutes, and 5 times.
7) Mu.l of substrate color development solution is added to each well, and the reaction is carried out at room temperature for 10-15 minutes in a dark place.
8) Mu.l of stop solution was added to each well and the Ig subclass was determined as a result of observation.
(8) ELISA identified the specificity of TFF1 monoclonal antibody 7G11. There are two important paralogues of TFF1, TFF2 and TFF3.TFF2 and TFF3 have similar functions and high protein homology with TFF1, and TFF2 and TFF3 proteins are prepared in the same way to identify whether the TFF1 monoclonal antibody prepared by the method can be in indiscriminate cross-bonding with TFF1, TFF2 and TFF3. The TFF1, TFF2 and TFF3 proteins were diluted to 5. Mu.g/ml with coating dilutions, respectively, and 100. Mu.l were added to each well of ELISA strips and coated overnight at 4 ℃. The next day the plate was removed and discarded and washed. TFF1 monoclonal antibody 7G11 was used as 1: 1000. 1: 2000. 1: 4000. 1: 8000. 1: 16000. 1: 32000. 1: 64000. 1:128000 diluted, 100 μl/well was added to the corresponding wells, and blank and yin-yang control wells were made. Incubation at 37 ℃ for 1 hour, plate washing, adding secondary antibody, 1:3000 HRP-labeled goat anti-mouse IgG was added to the corresponding wells at 100. Mu.l/well and incubated at 37℃for 40 min. The plate was discarded, washed, and dried by patting, 100. Mu.l/well of TMB substrate solution was added, developed in the dark for 10 minutes, and 50. Mu.l/well of stop solution was added.
The results are shown in Table 2, and the TFF1 monoclonal antibody 7G11 can better recognize TFF1, but not TFF1L, and has better specificity.
TABLE 2ELISA identification of specificity results of TFF1 monoclonal antibody 7G11
Example 2: in vivo effect evaluation of TFF1 monoclonal antibody on inhibiting transfer of breast cancer cells to bone cells caused by TFF1 comprises the following specific steps:
(1) And constructing a breast cancer bone metastasis model. Female nude mice of 6 weeks old initially develop a pre-breast cancer bone metastasis microenvironment after 20 days of tail vein injection of TFF1 (1 ng/kg once a day), and then a second breast pad of the nude mice is injected with a Luciferase-labeled breast cancer drug-resistant continuous state cell MCF-7-Luciferase DTP at an injection dose of 1×10 6 /only.
(2) TFF1 monoclonal antibodies inhibited breast cancer bone metastasis monitoring. TFF1 monoclonal antibody (0.1. Mu.g/kg, 1 time per day, 3 times total) or an equivalent amount of negative control mice lgG was injected into the mice of (1) via tail vein.
(3) Mice were examined for bone metastasis by a small animal biopsy imager and semi-quantitative studies were performed.
Specifically, fig. 1A-2B show schematic views of the in vitro inhibition of breast cancer cells by TFF1 monoclonal antibody 7G11 in fig. 1A-1B. FIG. 1A shows a TFF1 monoclonal antibody 7G11 treated group and FIG. 1B shows a negative control group.
Fig. 1C is a statistical plot of fig. 1A and 1B, showing that the breast cancer cell migration ability of the TFF1 monoclonal antibody 7G11 treated group was significantly inhibited.
FIGS. 2A and 2B are graphs showing the results of in vivo inhibition of breast cancer bone metastasis by TFF1 monoclonal antibody 7G11, which can be used to inhibit breast cancer bone metastasis.
Claims (10)
1. A hybridoma cell strain for preparing a TFF1 monoclonal antibody is characterized in that the preservation number is CCTCCNO: C2023397.
2. A TFF1 monoclonal antibody, characterized in that: is secreted by hybridoma cell strain with the preservation number of CCTCC NO: C2023397.
3. A method of preparing a TFF1 monoclonal antibody according to claim 1 or 2, comprising the steps of:
1) Designing and preparing a TFF1 immunogen polypeptide;
2) Immunizing a Balb/c mouse with the prepared immunogenic polypeptide;
3) The inguinal lymph node cells of the Balb/c mice in the step 2) and myeloma cells SP2/0 are subjected to multiple cell fusion by using a hybridoma technology, positive clones are screened by using an indirect ELISA method, and hybridoma cell strains capable of stably secreting the TFF1 monoclonal antibodies are screened in a cloning mode;
4) Preparing TFF1 monoclonal antibody ascites;
5) Purifying the TFF1 monoclonal antibody;
6) Identification of the TFF1 monoclonal antibody subclass;
7) Identification of the specificity of the TFF1 monoclonal antibody.
4. The method of producing a TFF1 monoclonal antibody according to claim 3, wherein step 1) comprises: the immunogenic polypeptide sequence site is designed in a region with better surface accessibility, flexibility, hydrophilicity and the like.
5. The method of preparing a TFF1 monoclonal antibody according to claim 3, wherein step 1) comprises: the designed immunogen polypeptide is TFF1-epitope69, and the corresponding polypeptide sequence site is: 69-82, the sequence of which is shown as SEQ ID NO. 1.
6. The method of preparing a TFF1 monoclonal antibody according to claim 3, wherein step 2) comprises: the Balb/c mice were 8 week old, female, normothermic mice.
7. The method of preparing a TFF1 monoclonal antibody according to claim 3, wherein step 3) comprises: performing primary immunization, secondary immunization, tertiary immunization and boosting immunization on the Balb/c mouse, wherein the primary immunization is to emulsify and mix the TFF1 immunogen polypeptide prepared in the step 1) with an equal volume of Freund's complete adjuvant to prepare immunogens, the secondary immunization and the tertiary immunization are to emulsify and mix the TFF1 immunogen polypeptide prepared in the step 1) with an equal volume of Freund's incomplete adjuvant to prepare immunogens, the boosting immunization is to emulsify and mix the TFF1 immunogen polypeptide prepared in the step 1) to prepare immunogens, and the dosage and the administration route of each immunogen are unchanged.
8. The method of preparing a TFF1 monoclonal antibody according to claim 3, wherein step 3) comprises: mixing inguinal lymph node separated cells with myeloma cells, using polyethylene glycol 1450 as fusion agent to form fusion cells, suspending the fusion cells in HAT culture solution containing calf serum, and juxtaposing 5% CO 2 The cells were cultured in a cell incubator at 37 ℃. Screening by the indirect ELISA method, wherein during screening, eukaryotic expression TFF1 is used for coating and screening, and the cell cloning is carried out on the cell growth holes of the detected positive clones by adopting a limiting dilution method until the culture solution of all the cell growth holes is positive.
9. The method of claim 3, wherein in step 6), the subset of TFF1 monoclonal antibodies is the Ig subset.
10. Use of the TFF1 monoclonal antibody of claim 1 or 2 in the manufacture of a medicament for treating breast cancer cell to bone cell conversion.
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