Anti-colorectal cancer polyclonal antibody, preparation method and application thereof
By preparing whole-cell tumor vaccines and combining them with traditional Chinese medicine using white oil adjuvant, high-titer polyclonal antibodies were obtained, solving the problems of complex preparation of monoclonal antibodies and poor efficacy of tumor vaccines, and achieving low-cost and high-efficiency treatment of colorectal cancer.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HENAN UNIV OF CHINESE MEDICINE
- Filing Date
- 2026-02-10
- Publication Date
- 2026-06-12
Abstract
Description
Technical Field
[0001] This invention relates to the fields of immunology and biotechnology, and in particular to a polyclonal antibody against colorectal cancer, its preparation method, and its application. Background Technology
[0002] Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of death. In my country, its incidence and mortality rates are on the rise. Clinical treatment primarily involves surgery, supplemented by radiotherapy and chemotherapy. These conservative treatments can prolong patient survival to some extent, but they also have significant side effects and a high recurrence rate, causing considerable physical and psychological suffering for patients. Therefore, finding more effective treatment methods is crucial to improving the treatment level of colorectal cancer and extending patient survival. Increasing evidence suggests that the immune system can prevent tumor growth, metastasis, and spread, and reduce the risk of recurrence and metastasis. Immunotherapy is an aggressive treatment approach that triggers the immune system to respond to tumor-specific antigens and attack tumor cells. Strategies include tumor-associated antigen peptides, intact tumor cells, in vitro generated dendritic cells (DCs) or tumor vaccines prepared using viruses as vectors, immune effector cells, genetically engineered T-cell receptors, and monoclonal antibodies. Although genetically modified cells have become a leader in immunotherapy, their complex preparation process and potential off-target effects have hindered their clinical advancement in treating solid tumors. Studies targeting cytokine-induced killer cells and natural killer cells in the treatment of solid tumors, including colorectal cancer, suggest that these treatments have some efficacy. However, due to a lack of antigen specificity, the reported clinical efficacy varies across different literatures. Furthermore, the identification of immunogenic tumor neoantigens remains challenging, time-consuming, and resource-intensive. Unresolved issues, such as whether these neoantigens can be effectively taken up by antigen-presenting cells through activating receptor pathways, have prevented current tumor vaccines from achieving the expected clinical efficacy. Therefore, the challenges of tumor immunotherapy in clinical practice remain.
[0003] my country began research and development of monoclonal antibodies in the 1980s, a relatively late start with a weak foundation. Internationally, monoclonal antibody drugs already hold the top position in the anti-tumor treatment market, while in China, traditional small-molecule drugs still dominate the anti-tumor drug market. Therefore, the domestic demand for antibody drugs is enormous. Monoclonal antibodies are typically prepared using hybridoma technology. While they offer advantages such as high purity, high sensitivity, strong specificity, and low cross-reactivity, their preparation is more complex, time-consuming, labor-intensive, and relatively expensive compared to polyclonal antibodies. Furthermore, epitopes are easily lost through chemical treatment of antigens, and the fixed affinity and limited biological activity of monoclonal antibodies restrict their application scope. Summary of the Invention
[0004] The purpose of this invention is to provide a polyclonal antibody against colorectal cancer, its preparation method, and its application, so as to solve the problems mentioned in the background art.
[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a polyclonal antibody against colorectal cancer, comprising the following steps:
[0006] (1) Human colorectal cancer HCT-116 cells were cultured in vitro in DMEM containing 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator. The culture medium was changed every 2-3 days, and the cells were passaged every 3-4 days. The cells were digested with trypsin for 1-5 minutes, collected, and carefully washed with PBS 2-3 times. The cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 50 μL of PBS. The cultured cells were then divided into 5×10⁶ cells / year groups. 6 / mL cell suspension, aseptic operation at 4℃;
[0007] (2) The cells cultured above were collected and divided into a non-inactivated group and an inactivated group. The non-inactivated group included a live cell group and high, medium and low concentration groups treated with mixed adjuvants. The cells in the inactivated group were inactivated by four different treatment methods to make them tumor vaccines that lost their carcinogenicity but retained their antigenicity.
[0008] (3) Mix white oil adjuvant, traditional Chinese medicine adjuvant and PBS in different proportions of (1-3):(1-3):(1-3) to form a mixed immune adjuvant. After mixing, stir with a magnetic stirrer for 5-10 minutes until it becomes a water-in-oil emulsion.
[0009] (4) Slowly drop the tumor seedlings prepared in step (2) into the adjuvant mixture prepared in step (3) using a micropipette. The ratio of tumor seedlings to adjuvant is 1: (1-3). Stir with a magnetic stirrer for 10-30 minutes while dropping the seedlings to ensure thorough mixing.
[0010] (5) Use the whole tumor cell vaccine for colorectal cancer prepared in step (4) as an immune antigen to immunize laying hens that are 2 months old and ready to lay. After the first immunization, the laying hens are immunized every 10 to 14 days by subcutaneous injection combined with intramuscular injection, for a total of 5 immunizations.
[0011] (6) Collect eggs laid by immunized laying hens every 10 to 14 days, separate egg white and egg yolk, extract yolk antibodies, purify them, and obtain polyclonal antibodies against colorectal cancer;
[0012] (7) Perform ELISA titer detection on the above polyclonal antibodies;
[0013] (8) In vitro experimental verification: MTT cell proliferation detection and TUNEL apoptosis detection;
[0014] (9) In vivo experimental verification: gross observation of tumors and in vivo imaging detection in small animals.
[0015] Preferably, the tumor cells cultured in step (1) include, but are not limited to, colorectal cancer cells.
[0016] Preferably, the tumor cells cultured in step (1) can be purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences or obtained from cancer tissues of clinical colorectal cancer patients through separation and primary culture of cancer cells.
[0017] Preferably, in step (2), the live cells are simultaneously treated with a mixed adjuvant at high, medium and low concentrations.
[0018] Preferably, the inactivated group treated by the four different methods in step (2) is consistent with the high concentration of live cells treated by the mixed adjuvant.
[0019] Preferably, a single adjuvant control group is set up simultaneously when immunizing animals in step (5).
[0020] Preferably, the specific steps in step (5) of immunizing laying hens with whole-cell colorectal cancer vaccine as an antigen are as follows:
[0021] The immunized animals were laying hens that were 2 months old at the time of their first laying.
[0022] The preferred sites for the subcutaneous multi-point injection are the neck, the base of the wings, the back, and the pectoralis major muscle.
[0023] The preferred site for intramuscular injection is the area on the outer thigh of the chicken where the muscles are most abundant.
[0024] The preferred injection dosage is 1 mL / chicken, 0.2 mL / site, with the initial immunization dose and subsequent booster immunization dose being the same;
[0025] The preferred method for multiple immunizations is immunization until the antibody titer reaches 2. 5 above.
[0026] Preferably, the specific procedures for extracting and purifying the egg yolk antibodies in step (6) are as follows:
[0027] Separate the egg yolk from the egg white, mix the PBS buffer with the egg yolk at a volume ratio of (2-10):1, stir well, and then add PEG6000 reagent to make the final concentration 1.5-6.0 wt%. Stir the mixture at 25-50℃ for 15-60 min, centrifuge at 2500-10000 rpm for 15-60 min, collect the supernatant, and obtain the egg yolk antibody, which is an anti-colorectal cancer polyclonal antibody.
[0028] A polyclonal antibody against colorectal cancer was prepared using the method described above.
[0029] Polyclonal antibodies against colorectal cancer can be used in the prevention and treatment of colorectal cancer. The specific steps are as follows:
[0030] (1) The polyclonal antibodies against colorectal cancer prepared above can be made into oral liquids, tablets, capsules, injections, etc., but not limited to the above dosage forms.
[0031] (2) The polyclonal antibody against colorectal cancer prepared above was used for in vitro and in vivo experimental efficacy testing:
[0032] ① Cell proliferation inhibition detection: Cells were seeded into 96-well plates. When the density reached 70% to 80%, polyclonal antibodies were added. After incubation for 24 h, 36 h, and 48 h, 20 μL of MTT solution was added directly to each well. After 4 h, the liquid was aspirated, 150 μL of LDMSO was added, and the plates were shaken at low speed for 10 min. The absorbance (A) value of each well was measured at 490 nm using a microplate reader.
[0033] ② Detection of apoptosis: After the cells from each group were crawled onto slides, they were washed once with PBS; the cells were fixed with 4% paraformaldehyde for 30-60 min; PBS containing 0.1% Tritium X-100 was added and the slides were incubated on ice for 2 min; 50 μL TUNEL detection solution was added and the slides were incubated at 37℃ in the dark for 60 min; the slides were then mounted with anti-fluorescence quenching mounting solution and observed under a confocal microscope.
[0034] ③ Tumor inhibition detection in nude mouse tumor model: A nude mouse colorectal cancer tumor model was established using firefly luciferase-labeled HCT-116-LUC cells. The mice were randomly divided into two groups: a PBS group and a multi-antibody treatment group, with 5 mice in each group. Under sterile conditions, intratumoral injection was performed, with an injection volume of 100 μL / mouse each time, once every three days for a total of 7 times. During the treatment period, the mice were weighed every 3 days to record changes in body weight, and the growth of the tumor was recorded using a small animal in vivo imaging system. The mice were sacrificed 30 days after the first administration, and the tumors were removed and photographed.
[0035] Compared with the prior art, the beneficial effects of the present invention are:
[0036] 1. This invention uses whole tumor cells as antigens to prepare tumor vaccines and uses them as immunogenic antigens to immunize laying hens. The resulting polyclonal antibodies can target multiple antigenic epitopes of tumor cells, resulting in good antibody efficacy and avoiding the adverse effects of monoclonal antibodies being prone to off-target effects.
[0037] 2. This invention compares the immune effects of live cell groups without adjuvant and those with adjuvant, and the results show that the role of adjuvant is indispensable in the process of live cell immunity.
[0038] 3. This invention uses white oil adjuvant to combine with traditional Chinese medicine for immune enhancement research. The results show that the combined traditional Chinese medicine has a better immune effect and can obtain high-titer polyclonal antibodies. A high-efficiency immunization program for preparing polyclonal antibodies against colorectal cancer has been successfully established for laying hens.
[0039] 4. This invention compared the time intervals of animal immunization. The antibody titer of egg yolk collected after the fourth booster immunization was the highest, while it decreased after the fifth immunization. This indicates that a suitable immunization cycle is also crucial for the preparation of polyclonal antibodies.
[0040] 5. Through multiple in vivo and in vitro experiments, this invention has found that the polyclonal antibody has a very significant anti-tumor effect in colorectal cancer experiments, and all results show statistical significance.
[0041] 6. The polyclonal antibody against colorectal cancer prepared by this invention has a simple preparation process and low cost. If combined with other anti-tumor products, it can be processed into granules, capsules, tablets, oral liquids, injections, etc. for the treatment of colorectal cancer. The preparation includes, but is not limited to, granules, capsules, tablets, oral liquids, injections, etc. Detailed Implementation
[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0043] Example 1
[0044] (1) Preparation of whole tumor cell antigen
[0045] ① Tumor cell culture: Human colorectal cancer HCT-116 cells were cultured in vitro in DMEM containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The culture medium was changed every 2-3 days, and the cells were passaged every 3-4 days. The cells were digested with trypsin for 1 min, collected, and carefully washed twice with PBS. The cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 50 μL of PBS. The cultured cells were then cultured to a density of 5 × 10⁶ cells / year. 6 Cell suspension, aseptic operation at 4°C.
[0046] ② Preparation of tumor vaccines: The cells cultured above were collected and divided into groups and inactivated using four different methods to make them tumor vaccines that lost their carcinogenicity but retained their antigenicity.
[0047] (2) Mix the white oil adjuvant and the traditional Chinese medicine compound adjuvant with PBS at a ratio of 1:1 and stir at high speed for 5 minutes with a magnetic stirrer until it becomes an oil-in-water state. Then slowly drip the tumor seed into the mixed adjuvant and stir at high speed for 10 minutes with a magnetic stirrer to mix thoroughly.
[0048] (3) The colorectal cancer whole tumor cell vaccine prepared above was used as an immunogen to immunize laying hens that were 2 months old and ready to lay eggs. After the initial immunization, the laying hens were immunized every 10 days by subcutaneous injection combined with intramuscular injection, for a total of 5 immunizations. The subcutaneous injection sites were the neck, the base of both wings, and the pectoralis major muscle. At the same time, an intramuscular injection was performed at the most abundant muscle on the outer thigh of one side of the chicken. Laying hens injected with PBS buffer were used as negative controls. The injection volume was 1 mL / chicken, 0.2 mL / site. The initial immunization dose and the subsequent booster immunization dose were the same, and a total of 4 booster immunizations were performed.
[0049] (4) Collect eggs laid by immunized laying hens every 10 days, separate egg white and egg yolk, extract egg yolk antibody, purify it to obtain polyclonal antibody against colorectal cancer; separate egg yolk from egg white, mix PBS buffer with egg yolk at a volume ratio of 2:1, stir evenly and add PEG6000 reagent to make the final concentration 1.5wt%; stir the mixture at 25℃ for 60min, centrifuge at 2500rpm for 60min, collect the supernatant to obtain egg yolk antibody, and store it at -20℃ for later use;
[0050] Example 2
[0051] (1) Preparation of whole tumor cell antigen
[0052] ① Tumor cell culture: Human colorectal cancer HCT-116 cells were cultured in vitro in DMEM containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The culture medium was changed every 2-3 days, and the cells were passaged every 3-4 days. The cells were digested with trypsin for 1 min, collected, and carefully washed twice with PBS. The cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 50 μL of PBS. The cultured cells were then cultured to a density of 5 × 10⁶ cells / year. 6 Cell suspension, aseptic operation at 4°C.
[0053] ② Preparation of tumor vaccines: The cells cultured above were collected and grouped into four different groups for inactivation treatment using four different methods to make them tumor vaccines that lose their carcinogenicity but retain their antigenicity.
[0054] (2) Mix the white oil adjuvant and the mixed adjuvant of traditional Chinese medicine with PBS at a ratio of 2:1 and stir at high speed with a magnetic stirrer for 10 minutes to form a water-in-oil state. Then slowly drip the tumor seed into the mixed adjuvant and stir at high speed with a magnetic stirrer for 20 minutes to mix thoroughly.
[0055] (3) The colorectal cancer whole tumor cell vaccine prepared above was used as an immunogen to immunize laying hens that were 2 months old and ready to lay eggs. After the initial immunization, the laying hens were immunized every 12 days by subcutaneous injection combined with intramuscular injection. A total of 5 immunizations were performed. The subcutaneous injection sites were the neck, the base of both wings, and the pectoralis major muscle. At the same time, an intramuscular injection was performed at the most abundant muscle on the outer thigh of one side of the chicken. Laying hens injected with PBS buffer were used as negative controls. The injection volume was 1 mL / chicken and 0.2 mL / site. The initial immunization dose and the subsequent booster immunization dose were the same. A total of 4 booster immunizations were performed.
[0056] (4) Collect eggs laid by immunized laying hens every 12 days, separate egg white and egg yolk, extract egg yolk antibody, purify, and obtain polyclonal antibody against colorectal cancer; separate egg yolk from egg white, mix PBS buffer and egg yolk at a volume ratio of 6:1, stir evenly and add PEG6000 reagent to make the final concentration 3wt%; stir the mixture at 37℃ for 40min, centrifuge at 5000rpm for 40min, collect the supernatant, obtain egg yolk antibody, and store at -20℃ for later use.
[0057] Example 3
[0058] (1) Preparation of whole tumor cell antigen
[0059] ① Tumor cell culture: Human colorectal cancer HCT-116 cells were cultured in vitro in DMEM containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The culture medium was changed every 2–3 days, and the cells were passaged every 3–4 days. The cells were digested with trypsin for 1 min, collected, and carefully washed twice with PBS. The cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 50 μL of PBS. The cultured cells were then cultured to a density of 5 × 10⁶ cells / year. 6 Cell suspension, aseptic operation at 4°C.
[0060] ② Preparation of tumor vaccines: The cells cultured above were collected and divided into groups and inactivated using four different methods to make them tumor vaccines that lost their carcinogenicity but retained their antigenicity.
[0061] (2) Mix the white oil adjuvant and the Chinese herbal medicine ginsenoside compound adjuvant with PBS at a ratio of 3:1 and stir at high speed with a magnetic stirrer for 15 minutes to form an oil-in-water state. Then slowly drip the tumor seed into the mixed adjuvant and stir at high speed with a magnetic stirrer for 30 minutes to mix thoroughly.
[0062] (3) The colorectal cancer whole tumor cell vaccine prepared above was used as an immunogen to immunize laying hens that were 2 months old and ready to lay eggs. After the initial immunization, the laying hens were immunized every 14 days by subcutaneous injection combined with intramuscular injection. A total of 5 immunizations were performed. The subcutaneous injection sites were the neck, the base of both wings, and the pectoralis major muscle. At the same time, an intramuscular injection was performed at the most abundant muscle on the outer thigh of one side of the chicken. Laying hens injected with PBS buffer were used as negative controls. The injection volume was 1 mL / chicken and 0.2 mL / site. The initial immunization dose and the subsequent booster immunization dose were the same. A total of 4 booster immunizations were performed.
[0063] (4) Collect eggs laid by immunized hens every 14 days, separate egg white and egg yolk, extract egg yolk antibody, purify it to obtain polyclonal antibody against colorectal cancer; separate egg yolk from egg white, mix PBS buffer with egg yolk at a volume ratio of 10:1, stir evenly and add PEG6000 reagent to make the final concentration 6.0wt%; stir the mixture at 50℃ for 15min, centrifuge at 10000rpm for 15min, collect the supernatant to obtain egg yolk antibody, and store it at -20℃ for later use.
[0064] Antibody titer detection examples
[0065] (1) The anti-colorectal cancer polyclonal antibody prepared in Example 1 was subjected to ELISA titer detection. The ELISA procedure was performed according to standard steps. After the colorimetric reaction was terminated, the absorbance at 450 nm was measured using a fully automated microplate reader. The main parameters are as follows:
[0066] ① Coating antigen: Collect 6×10⁶ HCT-116 cells of cultured colorectal cancer. 6 Wash the cells twice with PBS, resuspend them directly in 4 mL of coating buffer, sonicate for 2 min, add 100 μL to each well of a 96-well plate for coating, and set up 3 replicates. Seal with plastic wrap, incubate at 37°C for 1 h, and then incubate overnight at 4°C.
[0067] ② Washing: Take out the coated plate from the previous day, pour out the liquid in the wells, add 250μL of washing solution to each well using a pipette, repeat 4 times, and finally invert the reaction plate onto absorbent paper to drain the washing solution from the wells.
[0068] ③ Add 250 μL of the 5% skim milk powder prepared the day before as the sealing solution, and let it stand at room temperature for 1 hour. Wash with the same washing solution as in ②.
[0069] ④ Sample dilution: After removing the sample from the refrigerator, centrifuge at 10,000 rpm for 5 minutes to dilute the sample to two ratios: 1:100 (10 μL of original sample solution + 990 μL of ultrapure water) and 1:10,000 (10 μL of the above sample dilution solution + 990 μL of ultrapure water).
[0070] ⑤ Sample addition: Add 150 μL of the test sample diluted 1:10000 to each well. Simultaneously prepare a dilution control and incubate at 37°C for 2 hours. Wash with the same washing solution as in ②.
[0071] ⑥ Add enzyme-labeled secondary antibody IgG. Take 100 μL of the labeled secondary antibody diluted 1:2000 in each well and incubate at room temperature in the dark for 1 hour. Wash with the same washing buffer as in ②.
[0072] ⑦ Add substrate for color development: Mix 50 μL each of color development solution A and B, then add 100 μL to each well using a multi-channel pipette. After 15 min at room temperature in the dark, add 50 μL of stop solution to each well.
[0073] ⑧ Record the 450nm reading using a cell imaging microplate reader. Note that the reading should be completed within 30 minutes; the longer the time, the higher the background level. Assess the polyclonal antibody titer based on the OD value.
[0074] Antibody efficacy in vitro and in vivo experimental examples
[0075] (1) In vitro experimental verification: MTT cell proliferation detection and apoptosis detection;
[0076] ① MTT assay for cell proliferation: Cells were seeded into 96-well plates and incubated with polyclonal antibodies for 24h, 36h, and 48h. 20μL of MTT solution was added directly to each well. After 4h, the liquid was aspirated, and 150μL of LDMSO was added. The plates were shaken at low speed for 10min. The absorbance (A) value of each well was measured at 490nm using a microplate reader. The assay was repeated 3 times. Cell proliferation inhibition rate = (1 - A value of experimental group / A value of control group) × 100%.
[0077] ② Apoptosis detection: In this experiment, adherent cell slides were used as the detection object, and the TUNEL method was used to detect cell apoptosis. The specific steps are as follows: Take the cells from each group and wash them once with PBS; fix the cells with 4% paraformaldehyde for 30-60 min; wash them once with PBS; add PBS containing 0.1% Tritium X-100 and incubate on ice for 2 min; wash them twice with PBS; add 50 μL of TUNEL detection solution and incubate at 37ºC in the dark for 60 min (during incubation, pay attention to keeping the surrounding area moist with water-soaked paper or cotton wool to minimize the evaporation of TUNEL detection solution); wash them three times with PBS; mount the slides with anti-fluorescence quenching mounting medium and observe them under a confocal microscope.
[0078] (2) In vivo experimental verification: small animal live imaging detection
[0079] Compared to traditional methods such as palpation and tumor volume measurement, highly sensitive bioluminescence imaging technology can detect tumor growth and metastasis, as well as changes in tumor volume after drug treatment, thus more accurately determining the efficacy of drug treatment. This can significantly reduce experimental costs, eliminate errors caused by individual differences in mice, obtain more reliable data, and ensure experimental accuracy. A colorectal cancer tumor model was established using firefly luciferase-labeled HCT116-LUC cells and randomly divided into two groups: a PBS group and a multi-antibody treatment group, with 5 mice in each group. Under sterile conditions, intratumoral injection was performed, with each injection being 100 μL per mouse, once every three days for seven consecutive times. During the treatment period, the mice were weighed every three days to record changes in body weight, and tumor growth was recorded using a small animal in vivo imaging system. After three weeks, the tumor light signal was significantly reduced. Thirty days after the first administration, the mice were sacrificed, and the tumors were removed and photographed.
[0080] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a polyclonal antibody against colorectal cancer, characterized in that, Includes the following steps: (1) Culture colorectal cancer cells, collect cells and resuspend them to prepare cell suspension; (2) The cells obtained in step (1) are inactivated to obtain a tumor vaccine that loses its carcinogenicity but retains its antigenicity; (3) Mix the white oil adjuvant with the traditional Chinese medicine adjuvant and mix with PBS buffer, stirring to form a water-in-oil mixed immune adjuvant; (4) Mix the tumor vaccine obtained in step (2) with the mixed immune adjuvant obtained in step (3), stir to fully emulsify, and obtain the whole tumor cell tumor vaccine; (5) Use the whole tumor cell vaccine obtained in step (4) as an immune antigen to perform primary immunization and multiple booster immunizations on laying hens; (6) Collect eggs from immunized hens, separate the yolks, extract and purify the yolk antibodies to obtain polyclonal antibodies against colorectal cancer.
2. The method for preparing a polyclonal antibody against colorectal cancer according to claim 1, characterized in that, In step (1), the colorectal cancer cells are human colorectal cancer HCT-116 cells. The culture conditions are as follows: cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator, changing the culture medium every 2-3 days, passaged every 3-4 days, digesting the cells with trypsin for 1-5 minutes, collecting and carefully washing the cells 2-3 times with PBS, centrifuging at 1500 rpm for 5 minutes, discarding the supernatant, resuspending in 50 μL of PBS, and aseptically preparing the cultured cells into 5 × 10⁶ cells per cell line at 4°C. 6 / mL cell suspension.
3. The method for preparing a polyclonal antibody against colorectal cancer according to claim 1, characterized in that, In step (2), the inactivation treatment is carried out by physical or chemical methods, including but not limited to freeze-thaw method, irradiation method, mitomycin C treatment or formaldehyde fixation method.
4. The method for preparing a polyclonal antibody against colorectal cancer according to claim 1, characterized in that, In step (3), the white oil adjuvant and the mixed adjuvant of traditional Chinese medicine are mixed with PBS at a ratio of (1-3):1, and the stirring time is 5-10 minutes.
5. The method for preparing a polyclonal antibody against colorectal cancer according to claim 1, characterized in that, In step (4), the volume ratio of the tumor vaccine to the mixed immune adjuvant is 1:(1-3), and the stirring time is 10-30 minutes.
6. The method for preparing a polyclonal antibody against colorectal cancer according to claim 1, characterized in that, In step (5), the laying hens are laying hens that are 2 months old and have started laying. The immunization method is: subcutaneous injection combined with intramuscular injection. After the initial immunization, a booster immunization is performed every 10 to 14 days for a total of 5 immunizations. The injection dose is 1 mL / hen, of which 0.2 mL is injected at each injection site.
7. The method for preparing a polyclonal antibody against colorectal cancer according to claim 1, characterized in that, In step (6), the specific steps for extracting and purifying the egg yolk antibody are as follows: the egg yolk is separated from the egg white, PBS buffer is mixed with the egg yolk at a volume ratio of (2-10):1, and after stirring evenly, PEG6000 reagent is added to make the final concentration 1.5-6.0 wt%. The mixture is stirred at 25-50℃ for 15-60 min, centrifuged at 2500-10000 rpm for 15-60 min, and the supernatant is collected to obtain the egg yolk antibody, namely the anti-colorectal cancer polyclonal antibody.
8. A polyclonal antibody against colorectal cancer, characterized in that, It is prepared by the method for preparing an anti-colorectal cancer polyclonal antibody according to any one of claims 1 to 7.
9. The use of the anti-colorectal cancer polyclonal antibody according to claim 8 in the preparation of a medicament for the prevention and treatment of colorectal cancer.
10. A pharmaceutical composition for the prevention and treatment of colorectal cancer, characterized in that, The pharmaceutical composition comprises the anti-colorectal cancer polyclonal antibody of claim 8 and pharmaceutically acceptable excipients, and the dosage form of the pharmaceutical composition is an oral liquid, tablet, capsule or injection.