Method for purifying CA153 natural antigen

Abstract

The invention discloses a purification method of a CA153 natural antigen. The purification method comprises the following specific steps: unfreezing human colostrum, centrifuging and taking an intermediate layer; sequentially carrying out isoelectric precipitation, cold ethanol precipitation and CM ion exchange chromatography on the middle layer to obtain a CA153 antigen; the method is rich in raw material source, easy to obtain, simple and easy to implement, and the problem of low density gradient centrifugation yield is solved. Experiments show that the active content of the concentrated CA153 antigen reaches 30000 U / mL or above, the stability is good, the recovery rate reaches 60% or above (the recovery rate is improved by about 40% compared with that before improvement), and the requirements of an in-vitro diagnostic kit can be completely met.

Description

Technical Field

[0001] This invention belongs to the field of CA153 antigen extraction technology, and relates to a method for purifying CA153 antigen. Background Technology

[0002] CA153 antigen is a mucoid glycoprotein located on the cell membrane. It is a variant of the epithelial surface glycoprotein of mammary cells, with a molecular weight of 300-450 kDa. It includes a membrane region, an intracellular region, and a glycosyl-rich extracellular region. It is recognized by anti-human milk fat globule membrane antibodies 115D8 and DF3 and is present in various adenocarcinomas, such as breast cancer, lung cancer, ovarian cancer, and pancreatic cancer. During cell carcinogenesis, the activation of glycosyltransferases causes changes in cell surface carbohydrates, leading to an increase in CA carbohydrate antigens, which are released from the cancer cell membrane into the bloodstream. These antigens can be used as tumor markers for the auxiliary diagnosis of tumors, monitoring of treatment efficacy, and determination of metastasis and recurrence.

[0003] Currently, the main methods for preparing CA153 antigen include natural extraction and genetic engineering. Genetic engineering primarily utilizes signal peptides, tag protein genes, polypeptide sequences, and full-length gene sequences, cloning them into transient and stable expression plasmids, and then transfecting these plasmids into appropriate recipient cells for expression. This process is complex and requires high technical expertise and is costly. Natural extraction mainly involves extraction from human breast milk, where it is highly expressed in functionally active epithelial cells (primarily lactating mammary epithelium) and tumor tissues. Purification methods include cesium chloride gradient centrifugation combined with immunoaffinity purification, but this method yields low CA153 antigen recovery rates. Therefore, it is imperative to find a CA153 antigen purification method that is abundant in material sources, relatively simple to operate, practical, and has a high recovery rate. Summary of the Invention

[0004] The purpose of this invention is to provide a simple method for purifying CA153 natural antigen, which is rich in raw materials, low in cost, and has a high recovery rate.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for purifying the natural antigen CA153, the specific steps of which are as follows: Step S1: Take the human colostrum out of the -20℃ freezer, rinse it with running water to thaw it, and pour it into a centrifuge cup after thawing. Step S2: Centrifuge the thawed human colostrum at 10,000-15,000 rpm and 2-6°C for 20-40 minutes to remove the upper layer of oil and the lower layer of sediment to obtain the middle layer, i.e., skim milk. Step S3: Add hydrochloric acid solution to skim milk to adjust the pH of skim milk to 5.0, which is close to the isoelectric point of casein. Stir at room temperature for 20-40 min, centrifuge at 10000-15000 rpm and 2-6℃ for 20-40 min to remove casein and obtain supernatant, i.e., whey. Adjust the pH of whey to 7.2-7.4 with sodium hydroxide solution. Step S4: Transfer the whey into a beaker that has been pre-cooled in an ice-water mixture, add cold ethanol while stirring, and continue stirring for 20-40 minutes under ice bath conditions after the addition is complete. Step S5: Centrifuge at 10000~15000rpm and 2~6℃ for 20~40min, discard the supernatant, resuspend the precipitate in 0.01M phosphate buffer or Tris-HCl solution at pH 7.0~7.5, centrifuge and take the supernatant as crude extract. Step S6: Pass the crude extract through a CM ion exchange column. The target protein is mainly in the flow-through, and the main protein bound to the column is lactoferrin. Collect the flow-through, wash, filter, and concentrate to obtain the CA153 natural antigen. After concentration, the activity content of the CA153 natural antigen reaches more than 30,000 U / mL, with good stability and a recovery rate of more than 60%. Add preservatives and store at ≤-20℃.

[0006] Furthermore, in step S2, the upper layer of grease is removed by filtering with a double layer of 400-mesh nylon mesh.

[0007] Furthermore, the cold ethanol used in step S4 needs to be pre-cooled in a -20°C refrigerator for ≥24 hours, and the volume ratio of whey to cold ethanol is 1 / 3 to 1 / 2.

[0008] Furthermore, in step S6, the hollow fiber column or membrane used for concentration and washing has a molecular cutoff of 30 kDa.

[0009] Furthermore, in step S6, the buffer used for equilibration of the CM ion exchange column is 0.01M phosphate buffer or Tris-HCl solution at pH 7.0-7.5; the buffer used for eluting impurities is 0.01M PB + 0.5M NaCl at pH 7.0-7.5 or 0.01M Tris-HCl + 0.5M NaCl at pH 7.0-7.5; the filtration buffer is 0.01M PBS at pH 7.2-7.4; and the preservative used is sodium azide or P300, with an addition amount of 0.1wt%-0.2wt%.

[0010] The beneficial effects and advantages of this invention are as follows: The living material (human colostrum) of this invention is abundant and readily available. The CA153 natural antigen is purified by isoelectric point precipitation, cold ethanol precipitation, ion exchange chromatography and hollow fiber washing and concentration.

[0011] 1. The two largest types of proteins in human colostrum are casein and lactoferrin. Due to the high glycosylation of the CA153 antigen, its isoelectric point cannot be determined. However, the isoelectric point precipitation method can basically remove all casein. 2. Organic solvents such as ethanol have certain hydrophilicity and strong lipophilicity, which can improve the extraction effect for some proteins that are strongly bound to lipids or have many polar side chains in the molecule and are insoluble in water. 3. The elution buffer for CM ion exchange chromatography is 0.01M phosphate buffer or Tris-HCl solution with pH 7.0~7.5. Under these conditions, the target protein can be completely eluted, avoiding the loss of the target protein caused by multi-step chromatography. 4. After concentration and filtration using a 30KD hollow fiber column or membrane, the CA153 antigen preservation buffer is replaced with 0.01M PBS pH 7.2. After concentration, the CA153 activity content reaches over 30,000 U / mL, with good stability and a recovery rate of over 60% (about 40% higher than the previous recovery rate), which can fully meet the requirements of in vitro diagnostic kits. Detailed Implementation

[0012] The present invention will be described in detail below with reference to the scheme and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified. Example

[0013] A method for purifying the natural antigen of CA153 includes the following steps: Step S1: Take the human colostrum out of the -20℃ freezer, rinse it with running water to thaw it, and pour it into a centrifuge cup after thawing. Step S2: Centrifuge the thawed human colostrum at 10,000 rpm and 4°C for 30 min to remove the upper layer of oil and the lower layer of sediment to obtain the middle layer, i.e., skim milk. The upper layer of oil is removed by filtering with a double layer of 400-mesh nylon mesh. Step S3: Add hydrochloric acid solution to skim milk to adjust the pH of skim milk to 5.0, which is close to the isoelectric point of casein. Stir at room temperature for 30 min, centrifuge at 10000 rpm and 4℃ for 30 min to remove casein and obtain the supernatant, i.e., whey. Adjust the pH of whey to 7.2 with sodium hydroxide solution. Step S4: Transfer the whey into a beaker that has been pre-cooled in an ice-water mixture, and add cold ethanol while stirring. After the addition is complete, continue stirring for 30 minutes under ice bath conditions. The cold ethanol used must be pre-cooled in a -20°C refrigerator for ≥24 hours. The volume ratio of whey to cold ethanol added is 1 / 3. Step S5: Centrifuge at 10000 rpm and 4℃ for 30 min, discard the supernatant, resuspend the precipitate in 0.01M phosphate buffer at pH 7.0, centrifuge again and collect the supernatant as the crude extract. Step S6: The crude extract was passed through a CM Sepharose Fast Flow cation exchange column (30 mL column bed volume, XK26 / 20 column model; all chromatographic media and columns were purchased from Cytiva). The target protein was mainly in the flow-through, and lactoferrin was mainly bound to the column. The flow-through was collected, washed, filtered, and concentrated to obtain the CA153 natural antigen. Preservatives were added and stored at ≤-20℃. The concentration and washing were carried out using a hollow fiber column or membrane with a molecular cutoff of 30 KD. The buffer used for equilibration of the CM column was pH 7.0, 0.01 M phosphate buffer. The buffer used for eluting impurities was pH 7.0, 0.01 M PB + 0.5 M NaCl. The washing buffer was pH 7.2, 0.01 M PBS. The preservative used was P300, added at 0.1 wt%. Example

[0014] A method for purifying the natural antigen of CA153 includes the following steps: Step S1: Take the human colostrum out of the -20℃ freezer, rinse it with running water to thaw it, and pour it into a centrifuge cup after thawing. Step S2: Centrifuge the thawed human colostrum at 15000 rpm and 4℃ for 30 min to remove the upper layer of oil and the lower layer of sediment to obtain the middle layer, i.e., skim milk. The upper layer of oil is removed by filtering with a double layer of 400 mesh nylon mesh. Step S3: Add hydrochloric acid solution to skim milk to adjust the pH of skim milk to 5.0, which is close to the isoelectric point of casein. Stir at room temperature for 30 min, centrifuge at 15000 rpm and 4℃ for 30 min to remove casein and obtain the supernatant, i.e., whey. Adjust the pH of whey to 7.4 with sodium hydroxide solution. Step S4: Transfer the whey into a beaker that has been pre-cooled in an ice-water mixture, and add cold ethanol while stirring. After the addition is complete, continue stirring for 30 minutes under ice bath conditions. The cold ethanol used must be pre-cooled in a -20°C refrigerator for ≥24 hours. The volume ratio of whey to cold ethanol added is 1 / 2. Step S5: Centrifuge at 10000 rpm and 4℃ for 30 min, discard the supernatant, resuspend the precipitate in pH 7.0, 0.01M Tris-HCl buffer, centrifuge after resuspending and take the supernatant as crude extract. Step S6: Pass the crude extract through a CM Sepharose Fast Flow cation exchange column (30 ml column bed volume, XK26 / 20 column model; all chromatographic media and column were purchased from Cytiva). The target protein was mainly in the flow-through, and lactoferrin was mainly bound to the column. Collect the flow-through, wash, filter, and concentrate to obtain the CA153 natural antigen. Add preservative and store at ≤-20℃. The concentration and washing were carried out using a hollow fiber column or membrane with a molecular cutoff of 30 KD. The equilibration buffer for the CM ion exchange column was pH 7.5, 0.01 M Tris-HCl. The buffer used to elute other proteins was pH 7.0, 0.01 M Tris-HCl + 0.5 M NaCl. The washing buffer was pH 7.2, 0.01 M PBS. The preservative used was P300, added at 0.1 wt%.

[0015] Performance testing: 1. CA153 antigen content determination method: CA153 antigen detection luminescent kit.

[0016] 2. CA153 antigen stability assay: reconstitution stability at 2~8℃ and accelerated thermal stability at 37℃ for 14 days.

[0017] 3. Measurement Results 3.1 Results of CA153 content determination The CA153 natural antigen purified in Examples 1 and 2 was determined according to the above-described method for CA153 content determination. The results are shown in Table 1 below:

[0018] Conclusion: The content of natural CA153 antigen purified in Examples 1 and 2 was basically the same, and higher than that in the control.

[0019] 3.2 Stability test results The ferritin purified in Examples 1 and 2 was determined according to the enzyme stability assay method described above, and the stability results are shown in Table 2 below:

[0020] Note: In the table, 01 represents the control, 02 represents Example 1, and 03 represents Example 2; Q1 represents Level 1, Q2 represents Level 2, and Q3 represents Level 3.

[0021] As can be seen from the data in Table 2, the stability of the ferritin purified in Examples 1 and 2 decreased by less than 10% after being placed at 2℃-8℃ for 14 days and at 37℃ for 14 days.

[0022] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for purifying CA153 natural antigen, characterized in that... The specific steps are as follows: Step S1: Take the human colostrum out of the -20℃ freezer, rinse it with running water to thaw it, and pour it into a centrifuge cup after thawing. Step S2: Centrifuge the thawed human colostrum at 10,000-15,000 rpm and 2-6°C for 20-40 minutes to remove the upper layer of oil and the lower layer of sediment to obtain the middle layer, i.e., skim milk. Step S3: Add hydrochloric acid solution to skim milk to adjust the pH of skim milk to 5.0, which is close to the isoelectric point of casein. Stir at room temperature for 20-40 min, centrifuge at 10000-15000 rpm and 2-6℃ for 20-40 min to remove casein and obtain supernatant, i.e., whey. Adjust the pH of whey to 7.2-7.4 with sodium hydroxide solution. Step S4: Transfer the whey into a beaker that has been pre-cooled in an ice-water mixture, add cold ethanol while stirring, and continue stirring for 20-40 minutes under ice bath conditions after the addition is complete. Step S5: Centrifuge at 10000~15000rpm and 2~6℃ for 20~40min, discard the supernatant, resuspend the precipitate in 0.01M phosphate buffer or Tris-HCl solution at pH 7.0~7.5, centrifuge and take the supernatant as crude extract. Step S6: Pass the crude extract through a CM ion exchange column. The target protein is mainly in the flow-through, and the main protein bound to the column is lactoferrin. Collect the flow-through, wash, filter and concentrate to obtain the CA153 natural antigen. After concentration, the activity content of the CA153 natural antigen reaches more than 30,000 U / mL, with good stability and a recovery rate of more than 60%. Then add preservatives and store at ≤-20℃.

2. The purification method for CA153 natural antigen according to claim 1, characterized in that: In step S2, the upper layer of grease is removed by filtering with a double layer of 400-mesh nylon mesh.

3. The purification method for CA153 natural antigen according to claim 1, characterized in that: The cold ethanol used in step S4 needs to be pre-cooled in a -20°C refrigerator for ≥24 hours, and the volume ratio of whey to cold ethanol is 1 / 3 to 1 / 2.

4. The purification method for CA153 natural antigen according to claim 1, characterized in that: In step S6, the hollow fiber column or membrane pack used for concentration and filtration has a molecular cutoff of 30 kDa.

5. The purification method for CA153 natural antigen according to claim 1, characterized in that: In step S6, the buffer used for equilibration of the CM ion exchange column is 0.01M phosphate buffer or Tris-HCl solution at pH 7.0-7.5; the buffer used for eluting contaminating proteins is 0.01M PB + 0.5M NaCl solution at pH 7.0-7.5 or 0.01M Tris-HCl + 0.5M NaCl solution at pH 7.0-7.5; the filtration buffer is 0.01M PBS at pH 7.2-7.4; the preservative used is sodium azide or P300, with an addition amount of 0.1wt%-0.2wt%.

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