Preparation method and application of carbohydrate protein CA72-4
By combining low-serum adherence and serum-free suspension culture with drug stimulation, the time-consuming and labor-intensive problem of large-scale production of CA72-4 was solved, and efficient and economical large-scale production was achieved, with a significant increase in output.
Patent Information
- Application Number
- CN202410422545.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-09
- Publication Date
- 2025-10-14
AI Technical Summary
In the existing technology, the large-scale production of carbohydrate protein CA72-4 is time-consuming and labor-intensive, and the adherent culture method is not suitable for large-scale production.
Low-serum adherent culture and serum-free full suspension culture methods were used, combined with gemcitabine drug stimulation, and culture conditions were optimized to increase CA72-4 production, including subculture, suspension culture and concentration steps, to construct a serum-free full suspension culture LS-174T cell line.
The CA72-4 yield was more than 10 times higher than that of traditional adherent culture, which reduced production costs and contamination probability, made it suitable for large-scale production, and improved production efficiency and economic benefits.
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Figure CN120775941A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of in vitro diagnostic reagents, and specifically relates to a preparation method and application of a carbohydrate protein CA72-4. Background Art
[0002] LS-174T cells are human colon cancer cells. They are a trypsinized variant of the LS180 colon adenocarcinoma cell line and can produce large amounts of carcinoembryonic antigen (CEA). Electron microscopy reveals abundant microfilaments and cytoplasmic mucin vacuoles.
[0003] The CA72-4 antigen, extracted from LS-174T cells, is a mucin-like, high-molecular-weight glycoprotein with a molecular weight of 220-400 kDa, recognized by two monoclonal antibodies, cc49 and B72.3. CA72-4 is a biomarker for detecting gastric cancer and various digestive tract cancers. It is a chemical substance that reflects the presence of tumors. It is absent from normal adult tissues and only found in embryonic tissues. Normal serum levels are <6 U / mL, while abnormally elevated levels can be seen in various digestive tract tumors and ovarian cancer. It has high specificity for gastric cancer detection, with a cutoff value of >6 U / mL. Therefore, its presence or quantitative changes can be used to understand tumor histogenesis and cellular differentiation, aiding in diagnosis, prognosis, and treatment guidance.
[0004] Currently, CA72-4 is a large glycoprotein, making recombinant protein expression difficult. The market primarily focuses on natural CA72-4 antigens extracted from human colon cancer cells, LS-174T. Products such as Antibodies, Abbexa, and Daiao Biotechnology currently offer naturally extracted CA72-4 antigens. However, conventional LS-174T tumor cell culture requires adherent culture, making large-scale production time-consuming and labor-intensive. Summary of the Invention
[0005] The present invention discloses a preparation method and application of carbohydrate protein CA72-4, so as to solve the problem of time-consuming and labor-intensive mass production in the prior art.
[0006] In a first aspect of the present application, a method for preparing a carbohydrate protein CA72-4 is provided, comprising:
[0007] Adherent LS-174T cells were inoculated into DMEM medium containing 5% and 2.5% fetal bovine serum for subculture to obtain a low-serum adherent LS-174T cell line:
[0008] The low-serum adherent culture LS-174T cell line was inoculated into a serum-free suspension medium for subculture to obtain a serum-free full suspension culture LS-174T cell line;
[0009] The LS-174T cell line is cultured in suspension, and drugs are added in the process of suspension culture to stimulate the LS-174T cell line to secrete CA72-4.
[0010] The supernatant of the cells cultured for 3-5 days is concentrated to obtain concentrated CA72-4.
[0011] Optionally, the adherent LS-174T cells are inoculated into DMEM medium containing 5% or 2.5% fetal bovine serum for subculture, and the subculture is performed for at least 3 generations, the subculture ratio in DMEM medium with different concentrations of fetal bovine serum is 1:(2-5), and the subculture is performed at 37°C and 5% CO2.
[0012] Optionally, the low-serum adherent LS-174T cell line is inoculated into serum-free suspension culture medium for subculture, and the subculture is performed for at least 3 generations, each generation is cultured for 3-5 days, the subculture density is 0.5*10^6-1.0*10^6, and the subculture is performed at 37°C, 8% CO2, and 90-120 r / min.
[0013] Optionally, the drug added in the process of suspension culture is gemcitabine.
[0014] Optionally, the amount of the drug added in the process of suspension culture is greater than or equal to 200 ng / mL.
[0015] Optionally, the inoculation density of the suspension culture is 2.5*10^6-4*10^6, and the culture time is 3-5 days.
[0016] Optionally, before the adherent LS-174T cells are inoculated into DMEM medium containing 5% or 2.5% fetal bovine serum for subculture, the following steps are further included:
[0017] One adherent LS-174T cell is recovered, inoculated into DMEM medium, and cultured by adding 10% fetal bovine serum, and the culture conditions are 37°C, 5% CO2, and pH 7.2.
[0018] After the cells adhere and the monolayer confluence in the culture bottle reaches 95%, the culture solution is completely discarded, and the cells are subcultured for 3 generations by 0.25% EDTA-trypsin digestion and dispersion to obtain the adherent LS-174T cells for domestication.
[0019] In a second aspect, the application provides a use of CA72-4 prepared by the preparation method of any one of claims 1-7 in detecting the chemiluminescence activity of a sample.
[0020] The adherent cell culture process is complicated and not suitable for large-scale culture, and cannot meet the actual production needs. The serum-free whole-suspension culture LS-174T cell line constructed in the application can adapt to serum-free whole-suspension culture, facilitate the full contact between nutrients in the culture medium and cells, and high-density growth of cells, and reduce the difficulty of cell culture in the production process and the probability of contamination; in addition, it is beneficial to large-scale production in a short time, and the serum-free culture medium completely replaces the serum-containing culture medium, greatly reducing the production cost. The domesticated suspension culture cells provided in the application can produce CA72-4 with a yield higher than 10 times that of adherent culture, and can produce considerable economic benefits. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 Figure 3 is a morphological diagram of low serum adherent LS-174T cells in Example 3;
[0022] Figure 2 Figure 4 is a morphological diagram of serum-free whole-suspension culture LS-174T cells in Example 3. DETAILED DESCRIPTION
[0023] The technical solutions in the examples of the application will be described clearly and completely below. Obviously, the described examples are only a part of the examples of the application, rather than all the examples. Based on the examples in the application, all other examples obtained by those skilled in the art without creative labor belong to the scope of protection of the application.
[0024] Example 1
[0025] 1. Tumor drug class screening
[0026] 1) 9 drug classes and addition amount: 5-fluorouracil (200 ng / ml) / paclitaxel (10 ng / ml) / gemcitabine (50 ng / ml) / cisplatin (2 ug / ml) / camptothecin (10 ng / ml) / docetaxel (30 ng / ml) / gefitinib (25 ng / ml) / nilotinib (2 ug / ml) / irinotecan (5 ug / ml); the addition amount is determined according to the LD50 index of each drug on tumor cells;
[0027] 2) LS-174T cells are adherently cultured by using a traditional method, and 10 150 mm dishes are reached;
[0028] 3) When the cell confluence reaches 70-80%, the stimulating drug is added;
[0029] 4) After 3 days of drug stimulation culture, 1 mL of sample is taken, and the chemiluminescence activity of the sample is determined, that is, RLU (Relative Light Unit, relative light value). The larger the RLU value, the better the chemiluminescence activity.
[0030] Table 1 Effects of 9 stimulating drugs on the secretion of CA72-4 by adherent LS-174T cells
[0031]
[0032] In the above examples, the blank control refers to the absence of any stimulating drug. From the results in Table 1, it can be seen that the stimulating effect of gemcitabine is significantly higher than that of the control. Therefore, this application selects gemcitabine as the stimulating drug.
[0033] Example 2
[0034] 2. Optimization of drug-stimulated cell secretion conditions for CA72-4
[0035] 1) Drug concentration optimization:
[0036] a) Using conventional methods, culture 174T cells adherently to four 150 mm dishes.
[0037] b) When the cell confluence reached 70-80%, gemcitabine was added in a gradient to stimulate the cells to secrete CA72-4, with the concentrations reaching 0 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL, respectively;
[0038] c) After 3 days of drug stimulation, 1 mL of sample was collected and the chemiluminescence activity of the sample was measured.
[0039] Table 2 Effects of different stimulation concentrations on the secretion of CA72-4 by adherent LS-174T cells
[0040]
[0041] The optimal amount of gemcitabine added can increase the activity of CA72-4 in the cell supernatant by 5-10 times. As can be seen from Table 2, increasing the amount of gemcitabine added from 100 ng / mL to 200 ng / mL and 500 ng / mL has little effect on the activity of CA72-4 in the cell supernatant. Therefore, a concentration of 100 ng / mL can be selected for stimulation.
[0042] 2) Optimization of drug action time in 174T cells:
[0043] a) Culture 174T cells until they fill four 150 mm dishes;
[0044] b) Add the optimal stimulation concentration of 100 ng / mL at 0d / 1d / 2d / 3d / 4d after passage, and collect 1 ml of sample on 3d, 5d, and 6d after drug treatment to measure the chemiluminescence activity of the samples.
[0045] Table 3 Effects of different drug addition times on the secretion of CA72-4 by adherent LS-174T cells
[0046]
[0047]
[0048] As shown in Table 3, the activity of the samples did not increase significantly when the drug exposure time was extended from 3 days to 6 days. However, the activity of the samples increased significantly after 3 days of culture compared with 0-2 days after drug addition, and decreased slightly after 4 days. Therefore, 3 days after subculture was determined to be the optimal time for drug addition and the optimal drug exposure time was 3 days.
[0049] Example 3
[0050] 3. Construction of serum-free full suspension culture LS-174T cell line
[0051] 1) Resuscitate a single adherent LS-174T cell line and inoculate it into DMEM medium (Gibco product) supplemented with 10% fetal bovine serum (Hyclone product) at 37°C, 5% CO2, pH 7.2) containing 10% fetal bovine serum. Once the cells have adhered and the monolayer confluence in the culture flask reaches 95%, completely discard the culture medium and disperse the cells using 0.25% EDTA-trypsin. Subculture the cells for three generations according to conventional methods. Cells with clear edges, flat morphology, and normal growth can be used for acclimation.
[0052] 2) The adherent LS-174T cells obtained in step 1) were cultured in DMEM medium containing 5% and 2.5% fetal bovine serum for three generations, with the subculture ratio being 1:3 and the culture conditions being 37°C, 5% CO2, pH 7.2 to obtain a low serum adherent LS-174T cell line with cell morphology as shown in FIG. Figure 1 As shown;
[0053] 3) After the confluence of the last generation of low-serum adherent cell monolayer in step 2) reaches 95%, the culture medium is completely discarded, and the cells are dispersed using 0.25% EDTA-trypsin digestion. After counting the cells, the cells are directly diluted to a density of 1×10 6 cells / mL using serum-free full suspension medium; wherein the full suspension medium is OpMed medium (OPM-AM198V9, C231824);
[0054] 4) Cultivate for 5 generations at 37°C, 8% CO2, and 100 rpm shaking to obtain a serum-free, fully suspended LS-174T cell line;
[0055] 5) Suspension culture cells such as Figure 2There is agglomeration phenomenon, and the culture conditions can be continuously optimized.
[0056] 4. Suspension cultured LS-174T cells efficiently express native CA72-4
[0057] 1) Optimization of drug stimulation concentration for suspension cultured cells:
[0058] a) Dilute the acclimated serum-free suspension culture LS-174T cells to a density of 0.5 × 10^6 cells / mL and culture in 5 flasks (20 mL / flask). Culture conditions were 37°C, 8% CO2, and shaken at 100 rpm.
[0059] b) After 3 days of cell culture, gemcitabine was added in a gradient manner to stimulate the suspension cells to secrete CA72-4, with the concentrations reaching 0 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, and 500 ng / mL, respectively;
[0060] c) After 3 days of drug stimulation and culture, 1 mL of sample was collected from each cell to measure the chemiluminescent activity of CA72-4 in the cell secretion supernatant.
[0061] Table 4 Effects of different gemcitabine additions on the secretion of CA72-4 by suspension cells
[0062]
[0063] By testing the luminescence value of sample CA72-4, it was found that when the gemcitabine stimulation concentration of suspension culture cells was 200 ng / mL, the drug stimulation effect basically reached the highest point. If the drug stimulation concentration was continued to increase, the secretion supernatant activity tended to be stable.
[0064] 2) Optimization of cell seeding density in suspension culture:
[0065] a) Culture LS-174T cells in suspension at a seeding density of 1.5 x 10^6 / 2.5 x 10^6 / 4 x 10^6 in 3 flasks, 20 mL / flask;
[0066] b) Add 200 ng / mL gemcitabine on day 0 of inoculation to stimulate cell secretion of CA72-4;
[0067] c) Collect 1 ml of sample on the 3rd and 5th day after drug treatment and measure the chemiluminescence activity of the sample.
[0068] Table 5 Effect of CA72-4 secretion by suspension cells cultured at different inoculation densities
[0069]
[0070] As shown in Table 5, the activity of CA72-4 in the cell supernatant gradually increased with increasing seeding density. However, when the cell density increased from 2.5*10^6 to 4*10^6, the growth rate of CA72-4 activity decreased and no significant increase was observed. When the stimulation time was extended from 3 days to 5 days, the CA72-4 activity value increased by approximately 50%. Therefore, the culture conditions for high-secreting CA72-4 floating cells are: cell seeding density of 2.5-4*10^6, gemcitabine addition of 200ng / mL, and culture for 3-5 days.
[0071] 5. Purification and preservation of natural CA72-4 antigen
[0072] a) Concentration and medium replacement: The cell supernatant after 3-5 days of culture was concentrated and medium replaced using a membrane package (2513UBG294); wherein, the membrane package is an ultrafiltration membrane package manufactured by Hangzhou Jiuling Technology Co., Ltd., and the membrane package used in this example is part number G01100010K;
[0073] b) Concentration and exchange buffer: 20 mM PB, 50 mM NaCl pH 7.4;
[0074] c) Protein storage buffer: 20 mM PB, 50 mM NaCl pH 7.4, 10% glycerol.
[0075] The performance of the crude and pure CA72-4 antigen was tested as follows.
[0076] 1) Repeatability: The CV of the calibrator / quality control prepared from the crude CA72-4 antigen extracted according to the method of the present invention is less than 2%;
[0077] Table 6 Repeatability test results
[0078]
[0079] 2) Linearity: The crude purified CA72-4 antigen was diluted to the linear range of the kit. According to the following test data, the CA72-4 antigen obtained according to the present invention has excellent linearity, R 2 It can reach above 0.99;
[0080] Table 7 Linearity test results
[0081]
[0082]
[0083] 3) Stability: After 7 days at 37°C, the stability of CA72-4 crude antigen as a calibrator and quality control product decreased by less than 4%.
[0084] Table 8 Stability test results
[0085]
[0086] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are included in the scope of the claims of the present invention to be approved.
Claims
1. A method for preparing carbohydrate protein CA72-4, characterized in that: include: Adherent LS-174T cells were inoculated into DMEM medium containing 5% and 2.5% fetal bovine serum for subculture to obtain a low-serum adherent LS-174T cell line: The low-serum adherent culture LS-174T cell line was inoculated into a serum-free suspension medium for subculture to obtain a serum-free full suspension culture LS-174T cell line; LS-174T cell lines were cultured in suspension, and drugs were added during the suspension culture process to stimulate the LS-174T cell lines to secrete CA72-4; The cell supernatant after 3-5 days of culture was concentrated and the medium was replaced to obtain concentrated CA72-4.
2. The method for preparing carbohydrate protein CA72-4 according to claim 1, characterized in that: The adherent cultured LS-174T cells were inoculated into DMEM medium containing 5% and 2.5% fetal bovine serum for subculture. The number of subcultures was at least 3 generations. The subculture ratio in DMEM medium with different concentrations of fetal bovine serum was 1:(2-5). The subculture conditions were 37°C and 5% CO2.
3. The method for preparing carbohydrate protein CA72-4 according to claim 1, characterized in that: The low-serum adherent LS-174T cell line is inoculated into a serum-free suspension medium for subculture, with at least three subcultures, 3 to 5 days of culture per generation, a subculture density of 0.5*10^6 to 1.0*10^6, and subculture conditions of 37°C, 8% CO2, and 90 to 120 r / min.
4. The method for preparing carbohydrate protein CA72-4 according to claim 1, characterized in that: The drug added during suspension culture was gemcitabine.
5. The method for preparing carbohydrate protein CA72-4 according to claim 4, characterized in that: The amount of drug added during suspension culture is greater than or equal to 200 ng / mL.
6. The method for preparing carbohydrate protein CA72-4 according to claim 5, characterized in that: The inoculation density of suspension culture is 2.5*10^6~4*10^6, and the culture days are 3~5 days.
7. The method for preparing carbohydrate protein CA72-4 according to claim 1, characterized in that: Before the adherent cultured LS-174T cells are inoculated into DMEM medium containing 5% and 2.5% fetal bovine serum for subculture, the following is also included: A single adherent LS-174T cell line was revived and inoculated into DMEM medium supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and pH 7.
2. After the cells adhered to the wall and the monolayer confluence in the culture flask reached 95%, the culture medium was completely discarded, and the cells were digested and dispersed with 0.25% EDTA-trypsin. The cells were subcultured for three generations to obtain adherent culture LS-174T cells for acclimation.
8. Use of CA72-4 prepared by the preparation method according to any one of claims 1 to 7 in detecting the chemiluminescence activity of a sample.