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CN-122011152-A - Purification method of CA153 natural antigen

CN122011152ACN 122011152 ACN122011152 ACN 122011152ACN-122011152-A

Abstract

The invention discloses a purification method of a CA153 natural antigen, which comprises the specific steps of thawing human colostrum, centrifuging to obtain an intermediate layer, sequentially performing isoelectric point precipitation, cold ethanol precipitation and CM ion exchange chromatography on the intermediate layer to obtain the CA153 antigen. Experiments show that the activity content of the concentrated CA153 antigen reaches more than 30000U/mL, the stability is good, the recovery rate reaches more than 60 percent (about 40 percent higher than the recovery rate before improvement), and the requirements of an in-vitro diagnostic kit can be completely met.

Inventors

  • CAO XIAONAN
  • Tian Linqiang
  • LIU JIE
  • SUN ZHENZHOU
  • GU Yu

Assignees

  • 河南医药大学第三附属医院

Dates

Publication Date
20260512
Application Date
20260127

Claims (5)

  1. 1. A purification method of a CA153 natural antigen is characterized by comprising the following specific steps: Step S1, taking out human colostrum from a refrigerator at the temperature of-20 ℃, flushing with flowing water for thawing, and pouring the human colostrum into a centrifugal cup after thawing; Step S2, centrifuging the unfrozen human colostrum at 10000-15000 rpm and 2-6 ℃ for 20-40 min, and removing upper grease and lower sediment to obtain middle layer, namely skimmed milk; Step S3, adding hydrochloric acid solution into skim milk to adjust the pH value of the skim milk to be 5.0, approaching the isoelectric point of casein, stirring for 20-40 min at room temperature, centrifuging for 20-40 min at 10000-15000 rpm and 2-6 ℃, removing casein to obtain supernatant, namely whey, and adjusting the pH value of the whey to be 7.2-7.4 by using sodium hydroxide solution; S4, transferring the whey into a beaker which is cooled in an ice-water mixture in advance, adding cold ethanol while stirring, and continuing stirring for 20-40 min under an ice bath condition after the addition is finished; Step S5, centrifuging for 20-40 min at 10000-15000 rpm and 2-6 ℃, discarding the supernatant, re-suspending the precipitate by using a phosphate buffer solution or Tris-HCl solution with the pH of 7.0-7.5 and 0.01M, and centrifuging to obtain the supernatant, namely a crude extract; And S6, passing the crude extract through a CM ion exchange column, wherein the target protein is mainly in flow-through, and is mainly lactoferrin combined on a chromatographic column, collecting flow-through liquid, washing, filtering and concentrating to obtain the CA153 natural antigen, wherein the activity content of the concentrated CA153 natural antigen is more than 30000U/mL, the stability is good, the recovery rate is more than 60%, and then preservative is added for preservation at the temperature of less than or equal to-20 ℃.
  2. 2. The method for purifying a CA153 natural antigen according to claim 1, wherein the upper layer of fat is removed in the step S2 by filtration through a double-layer 400 mesh nylon mesh.
  3. 3. The method for purifying a CA153 natural antigen according to claim 1, wherein the cold ethanol used in the step S4 is pre-cooled for more than or equal to 24 hours in a refrigerator at-20 ℃, and the volume ratio of the added whey to the cold ethanol is 1/3-1/2.
  4. 4. The method of claim 1, wherein the hollow fiber column or membrane pack for concentration and filtration in step S6 has a molecular retention of 30KD.
  5. 5. The method for purifying a CA153 natural antigen according to claim 1, wherein the buffer solution used for CM ion exchange column balancing in the step S6 is a phosphate buffer solution or Tris-HCl solution with the pH of 7.0-7.5 and 0.01M, the buffer solution used for eluting the hybrid protein is a solution with the pH of 7.0-7.5 and 0.01M PB+0.5M NaCl or a solution with the pH of 7.0-7.5 and 0.01M Tris-HC1+0.5M NaCl, the washing buffer solution is a solution with the pH of 7.2-7.4 and 0.01M PBS, and the preservative is sodium azide or P300, and the addition amount of the preservative is 0.1-0.2 wt%.

Description

Purification method of CA153 natural antigen Technical Field The invention belongs to the technical field of extraction of CA153 antigen, and relates to a purification method of CA153 antigen. Background The CA153 antigen is a mucus-like glycoprotein located on cell membranes, is a variant of mammary gland cell epithelial surface glycoprotein, has a molecular weight of 300-450 KD, comprises a membrane region, an intracellular region and a glycosyl-rich extracellular region, is recognized by anti-human milk fat globule membrane antibodies 115D8 and DF3, and exists in various adenocarcinomas such as breast cancer, lung cancer, ovarian cancer, pancreatic cancer and the like. When the cells are cancerous, the glycosyl converting enzyme is activated to cause the change of cell surface saccharides, CA saccharide antigens are increased and separated from cancer cell membranes, and the CA saccharide antigens are released into blood, so that the CA saccharide antigens can be used as tumor markers for auxiliary diagnosis, curative effect monitoring and metastasis and recurrence judgment of tumors. The current preparation method of CA153 antigen mainly comprises natural extraction and genetic engineering preparation methods. The genetic engineering preparation method mainly utilizes signal peptide, tag protein gene, polypeptide sequence and full-length gene sequence, clones the signal peptide, tag protein gene, polypeptide sequence and full-length gene sequence into transient expression plasmid and stable expression plasmid, and transfects the plasmid into proper receptor cells for expression. The natural extraction is mainly extracted from human emulsion, and is expressed in high level in epithelium (mainly mammary gland epithelium in lactation period) and tumor tissue with active function, and the purification method comprises cesium chloride gradient centrifugation combined with immunoaffinity purification, but the recovery rate of CA153 antigen obtained by the method is not high. Therefore, it is imperative to find a CA153 antigen purification method which is rich in material source, relatively simple in operation, practical and high in recovery rate. Disclosure of Invention The invention aims to provide a purification method of CA153 natural antigen, which has the advantages of simple method, abundant raw material sources, low cost and high recovery rate. In order to achieve the above purpose, the invention adopts the following technical scheme: a purification method of CA153 natural antigen comprises the following specific steps: Step S1, taking out human colostrum from a refrigerator at the temperature of-20 ℃, flushing with flowing water for thawing, and pouring the human colostrum into a centrifugal cup after thawing; Step S2, centrifuging the unfrozen human colostrum at 10000-15000 rpm and 2-6 ℃ for 20-40 min, and removing upper grease and lower sediment to obtain middle layer, namely skimmed milk; Step S3, adding hydrochloric acid solution into skim milk to adjust the pH value of the skim milk to be 5.0, approaching the isoelectric point of casein, stirring for 20-40 min at room temperature, centrifuging for 20-40 min at 10000-15000 rpm and 2-6 ℃, removing casein to obtain supernatant, namely whey, and adjusting the pH value of the whey to be 7.2-7.4 by using sodium hydroxide solution; S4, transferring the whey into a beaker which is cooled in an ice-water mixture in advance, adding cold ethanol while stirring, and continuing stirring for 20-40 min under an ice bath condition after the addition is finished; Step S5, centrifuging for 20-40 min at 10000-15000 rpm and 2-6 ℃, discarding the supernatant, re-suspending the precipitate by using a phosphate buffer solution or Tris-HCl solution with the pH of 7.0-7.5 and 0.01M, and centrifuging to obtain the supernatant, namely a crude extract; And S6, passing the crude extract through a CM ion exchange column, wherein the target protein is mainly in flow-through, and is mainly lactoferrin combined on a chromatographic column, collecting flow-through liquid, washing, filtering and concentrating to obtain the CA153 natural antigen, wherein the activity content of the concentrated CA153 natural antigen is more than 30000U/mL, the stability is good, the recovery rate is more than 60%, and the preservative is added for preservation at the temperature of less than or equal to-20 ℃. Further, in the step S2, the upper layer grease is removed by a double-layer 400-mesh nylon net for filtering. Further, the cold ethanol used in the step S4 needs to be precooled in a refrigerator at the temperature of minus 20 ℃ for more than or equal to 24 hours, and the volume ratio of the whey to the cold ethanol is 1/3-1/2. Further, in step S6, the hollow fiber column or the membrane package for concentration and filtration is used, and the molecular cut-off amount is 30KD. Further, in the step S6, the buffer solution used for balancing the CM ion exchange column is a phosphoric acid buff