CN107698675A - Separate and purify the method and kit of calprotectin - Google Patents
Separate and purify the method and kit of calprotectin Download PDFInfo
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- 102000001109 Leukocyte L1 Antigen Complex Human genes 0.000 title claims abstract description 99
- 108010069316 Leukocyte L1 Antigen Complex Proteins 0.000 title claims abstract description 99
- 238000000034 method Methods 0.000 title claims abstract description 52
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 37
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 30
- 239000000287 crude extract Substances 0.000 claims abstract description 22
- 239000003480 eluent Substances 0.000 claims abstract description 20
- 210000000265 leukocyte Anatomy 0.000 claims abstract description 15
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 13
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims abstract description 13
- 238000000502 dialysis Methods 0.000 claims abstract description 12
- 239000012535 impurity Substances 0.000 claims abstract description 11
- 239000012460 protein solution Substances 0.000 claims abstract description 11
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 7
- 239000012149 elution buffer Substances 0.000 claims description 23
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 19
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 238000004140 cleaning Methods 0.000 claims description 16
- 210000004027 cell Anatomy 0.000 claims description 15
- 239000007853 buffer solution Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 12
- 210000004369 blood Anatomy 0.000 claims description 11
- 239000008280 blood Substances 0.000 claims description 11
- 239000000284 extract Substances 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 8
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- 239000002808 molecular sieve Substances 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 4
- 238000005342 ion exchange Methods 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 16
- 238000002360 preparation method Methods 0.000 abstract description 5
- 238000010339 medical test Methods 0.000 abstract description 3
- 238000001742 protein purification Methods 0.000 abstract description 2
- 229920000642 polymer Polymers 0.000 abstract 1
- 239000013256 coordination polymer Substances 0.000 description 16
- 238000000746 purification Methods 0.000 description 9
- 238000001155 isoelectric focusing Methods 0.000 description 8
- 239000000523 sample Substances 0.000 description 7
- 238000005571 anion exchange chromatography Methods 0.000 description 6
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 210000000440 neutrophil Anatomy 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- 102000014823 calbindin Human genes 0.000 description 5
- 108060001061 calbindin Proteins 0.000 description 5
- 210000003743 erythrocyte Anatomy 0.000 description 5
- 206010061218 Inflammation Diseases 0.000 description 4
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 208000002551 irritable bowel syndrome Diseases 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 108010052500 Calgranulin A Proteins 0.000 description 3
- 108010052495 Calgranulin B Proteins 0.000 description 3
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 102100032442 Protein S100-A8 Human genes 0.000 description 3
- 102100032420 Protein S100-A9 Human genes 0.000 description 3
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
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- 235000013601 eggs Nutrition 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
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- 241000894006 Bacteria Species 0.000 description 2
- 208000004232 Enteritis Diseases 0.000 description 2
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- 238000012512 characterization method Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 239000000815 hypotonic solution Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 1
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 206010022678 Intestinal infections Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000005935 Sulfuryl fluoride Substances 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000008004 cell lysis buffer Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000003145 cytotoxic factor Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000004565 granule cell Anatomy 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000004493 neutrocyte Anatomy 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- OBTWBSRJZRCYQV-UHFFFAOYSA-N sulfuryl difluoride Chemical compound FS(F)(=O)=O OBTWBSRJZRCYQV-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4728—Calcium binding proteins, e.g. calmodulin
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to Protein purification techniques field, and in particular to a kind of to separate and purify the method and kit of calprotectin, this method includes：Obtain leukocyte samples；Leukocyte samples are handled through clasmatosis to obtain endochylema crude extract；Ammonium sulfate is added into endochylema crude extract and obtains the first refined solution to remove impurity protein；Protein solution is obtained through the first refined solution described in dialysis treatment；The protein solution is handled through ion-exchange chromatography to obtain eluent；Eluent is handled through sieve chromatography to obtain the second eluent.The separation of the present invention simultaneously purifies the method for simplifying that the method for calprotectin is removed foreign protein, be combined using dialysis, ion-exchange chromatography and sieve chromatography by saltouing, calprotectin separation yield and purity is improved, and maintains the structural intergrity of calprotectin polymer to ensure the due accuracy in medical test；The present invention method and step it is simple, eliminate complexity etc. point focusing step, improve preparation efficiency.
Description
Technical field
The present invention relates to Protein purification techniques field, and in particular to a kind of to separate and purify the method for calprotectin and use
The method of stating separates and purifies the kit of calprotectin.
Background technology
People's calprotectin（Calprotectin, CP）Be the main cells of human leukocytes, neutrophil cell it is main
Plasmosin, it is that human body is inflamed the important of disease and other inflammatory reactions caused by bacterium or viral infection, tumour etc.
Identify molecule.The calprotectin levels in the body fluid such as blood, cerebrospinal fluid, urine, excrement or excreta are examined, can be with adjuvant clinical
The various inflammatory diseases of diagnosis；The calprotectin levels wherein detected in excrement are to discriminate between organic, inflammatory bowel disease（IBD,
Inflammatory Bowel Diseases）With functional disorder, non-inflammatory IBS（IBS, Irritable
Bowel Syndrome）Important indicator.
It is the standard side that intestines problem is examined that calprotectin/CP levels in fecal specimens are examined using immunologic detection method
Method, detecting its content exactly needs calprotectin/CP of natural origin as standard items and competition detection sample, therefore can
Largely, the people's calprotectin/CP for stably preparing high-purity is the premise of medical diagnosis enteritis.On the other hand, prepare special
Heterogenetic antibody is also required to obtain calprotectin/CP of substantial amounts of high-purity.
Currently, people's calprotectin/CP preparation flow is from people's blood using the traditional scheme in 1980-1990 ages
Separate, purify in liquid leucocyte；Including separation human leukocytes, cracking human leukocytes obtain full cell crude extract, by etc. voltolisation
Burnt and chromatography is separated, purified for the method for key step.Because the full cell crude extract of leucocyte contains largely
Foreign protein（Including other calbindins）, past separation, purification process continue to use chromatography and isoelectric focusing technique always
With reference to method carry out, not only complex steps, also need to try using specific instrument and isoelectric focusing using isoelectric focusing method
Agent, it is expensive, and have substantial amounts of loss, yield is severely impacted.On the other hand, etc. the method for point focusing protein isolate
Destroy calprotectin multimeric structure, the method that the albumen of acquisition is remixed, reconfigured, it is impossible to correctly reflect that endogenous calcium is defended
The situation of albumen.The biochemical characteristic of this method application calprotectin, foreign protein and the chromatography simplified step are removed by saltouing
Suddenly, complete high-purity calprotectin/CP can be efficiently obtained, yield and yield are all greatly enhanced.
People's calprotectin（Calprotectin , CP）It is molecular weight about 22-36KD protein multimers molecule (master
To be heterodimer or tripolymer), including calbindin S100A8（14KD）With calbindin S100A9（8KD）Two hatching eggs
White molecular components, the neutrophil cell and monocyte that are primarily present in human blood etc., neutrophil cell endochylema can be accounted for
The 60% of albumen, i.e., each neutrophil leucocyte containing up to 25pg calprotectin/CP, with red blood cell in hemoglobin contain
Amount（30pg）It is equally matched.In inflammation disease and because in inflammatory reaction caused by bacterium, virus infection and tumour etc., neutrophil(e) granule
Cell is activated, substantial amounts of calprotectin/CP can be discharged into various body fluid.In enteric infection, parasitic disease, inflammatory bowel
The diseases such as inflammation, intestinal canal tumour occur when neutrophil migration to intestinal mucosa, can largely discharge calprotectin into enteric cavity,
Therefore in the excrement of the patients such as enteritis can detect due to the releases such as intestinal mucosal injury neutrophil cell calprotectin/
CP。
People's calprotectin/CP immune detection needs native protein standard items and competition test sample, with to patient samples
Quantitative testing is carried out, and avoids false positive；On the other hand, the antibody that specificity is directed to natural calprotectin/CP is prepared, it is also desirable to
Calprotectin/CP of high-purity is largely separated, is purified into from human blood, and this has very big technical difficulty.
The method of sponsor's calprotectin in past document/CP purifying mainly using chromatography with etc. two kinds of point focusing
The strategy that technology is combined, initial crude protein liquid is used as using the full cell pyrolysis liquid of blood leucocyte.Due to full cell pyrolysis liquid
Contain substantial amounts of cell foreign protein（Including calbindin）, therefore employ point that chromatography is combined with isoelectric focusing
From, purification step.It is expensive etc. the method not only complex steps, and need to use specific instrument and reagent of point focusing,
Yield and yield are all affected；The structure of protein multimers is also destroyed simultaneously, and influence is subsequently used for the accurate of medical test
Property.
Nineteen eighty-three, Dale I. et al. describe a large amount of separation, the method for Purification of Human calprotectin first, including prepare white
Cell coarse body fluid, etc. point focusing and dialysis or step, the yield such as sieve chromatography be about 20%（Dialysis）It is or lower（Using molecule
Sieve chromatography method）（Purification and partial characterization of a highly immunogenic
human leukocyte protein, the L1 antigen, Eur. J. Biochem. 134:1-6）.
Nineteen ninety-five, Yui S. et al. report separation, the purification process of rat calprotectin, including ammonium sulfate precipitation,
The steps such as sieve chromatography, isoelectric focusing and anion-exchange chromatography（Purification and characterization
of the cytotoxic factor in rat peritoneal exudate cells: it identification as
the calcium binding protein complex, calprotectin。 J. Leukoc. Biol. 58: 307-
316）.
1998, van den Bos C. et al. report two step isolation and purification methods of people's calprotectin, using the moon
Ion-exchange chromatography chromatography goes out the mixture of three kinds of calbindins（Two subunit MRP8 of P6 and calprotectin and
MRP14）, then P6 is separated with calprotectin by the method for isoelectric focusing again（Copurification of P6, MRP8,
and MRP14 from Human Granulocytes and Separation of Individual Proteins.
Protein Expression and Purification 13(3):313-8）.
Currently, calprotectin/CP preparation flow continues to use the traditional scheme in 1980-90 ages substantially, main to include from people
Or the full cell crude extract that obtains of other animal blood write cell lysis buffers by chromatography and etc. the method for point focusing carry out
Separation, purifying, because the full cell crude extract of leucocyte contains substantial amounts of foreign protein, required chromatography and isoelectric focusing side
Method complex steps, it is costly and have substantial amounts of loss；The structure of protein multimers and complete is destroyed using isoelectric focusing method
Property, for being affected as the accuracy of protein standard substance and competition sample.
In consideration of it, the defects of overcoming the above in the prior art, there is provided a kind of method for separating and purifying calprotectin turns into
This area technical problem urgently to be resolved hurrily.
The content of the invention
It is an object of the invention to the drawbacks described above for prior art, there is provided a kind of to separate and purify the side of calprotectin
Method and a kind of separation simultaneously purify the kit of calprotectin.
The purpose of the present invention can be realized by following technical measures：
A kind of method for separating and purifying calprotectin, comprises the following steps：
a）Obtain the leukocyte samples for including the calprotectin；
b）The leukocyte samples are handled through clasmatosis to obtain the endochylema crude extract comprising the calprotectin；
c）Ammonium sulfate is added into endochylema crude extract and obtains the first purifying comprising the calprotectin to remove impurity protein
Liquid；
d）The protein solution for including the calprotectin is obtained through the first refined solution described in dialysis treatment at least once；
e）The protein solution is handled through ion-exchange chromatography at least once to obtain the eluent for including the calprotectin；With
f）The eluent is handled through sieve chromatography to obtain the second eluent comprising calprotectin.
Preferably, in step b）Afterwards, step c）Also comprise the following steps before：
EDTA is added into endochylema crude extract slightly to carry to obtain the endochylema comprising the calprotectin with aids precipitation impurity protein
Liquid.
Preferably, in step d）In, the pH of elution buffer is 8.0~8.5, and Tris-HCl concentration is in elution buffer
15~25mmol/L, EDTA concentration are 4.0~6.0mmol/L.
Preferably, in step e）In, the protein solution is subjected to Image processing, Ran Houyong by ion exchange column first
Cleaning buffer solution is cleaned, and the eluent for including the calprotectin is finally eluted and collected with elution buffer；Its
In, the pH of the cleaning buffer solution is 8.0~8.5, and Tris-HCl concentration is that 15~25mmol/L, EDTA are dense in cleaning buffer solution
Spend for 4.0~6.0mmol/L；The pH of the elution buffer is 8.0~8.5, and Tris-HCl concentration is 15 in elution buffer
~25mmol/L, EDTA concentration is 4.0~6.0mmol/L.
Preferably, ammonium sulfate concentrations are 35% w/v.
Preferably, the step a）The leukocyte samples for including the calprotectin are specially obtained from people's whole blood.
Preferably, first refined solution further carries out filtration step.
Preferably, in step b）In, crush leucocyte with homogenizer and obtain endochylema crude extract, the pH of plasmosin extract
For 6.5~7.0, in plasmosin extract Tris-HCl concentration be 15~25mmol/L, NaCl concentration be 1.5~2.5mmol/
L, MgCl2 concentration is 3.0~4.0 mmol/L.
Present invention also offers a kind of separation and the kit of calprotectin is purified, including：
For extracting the plasmosin extract of cell protein；
For removing the ammonium sulfate of impurity protein；
Elution buffer for dialysis treatment；
For cleaning the cleaning buffer solution of chromatographic column；
Elution buffer for elution chromatography post；With
For eluting the second elution buffer of molecular sieve.
Preferably, the kit also includes：For aiding in removing deimpurity EDTA solution.
The separation of the present invention simultaneously purifies the method for calprotectin and removes foreign protein, using dialysis, ion exchange by saltouing
The method for simplifying that chromatography and sieve chromatography are combined, calprotectin separation yield and purity greatly improved, and maintain calcium
The structural intergrities of protein multimers is defended to ensure the due accuracy in medical test；The method and step of the present invention is simple,
Eliminate complexity etc. point focusing step, improve preparation efficiency, it is easy to utilize.
Brief description of the drawings
Fig. 1 is the anion-exchange chromatography result figure of embodiments of the invention 1.Anion exchange chromatography result, group
Divide the calprotectin that 9-15 is enrichment.
Fig. 2 is the protein electrophoresis figure of the eluent after the anion-exchange chromatography of embodiments of the invention 1.SDS-PAGE eggs
Leucismus gel electrophoresis, coomassie brilliant blue staining, show the calprotectin of enrichment（Component 9-15）.
Fig. 3 is the sieve chromatography result figure of embodiments of the invention 1.Sieve chromatography result：Show highly enriched, pure
The calprotectin component of change.
Fig. 4 is the protein electrophoresis figure of the eluent after the sieve chromatography of embodiments of the invention 1.SDS-PAGE albumen becomes
Property gel electrophoresis, coomassie brilliant blue staining, show the calprotectin of purifying（Component 26-31）.
Fig. 5 is the calprotectin purification effect figure of embodiments of the invention 1.Protein Marker（KD）, the calcium of purifying
Defend albumen and include two polypeptide chains, molecular weight is respectively 8KD and 14KD.
Fig. 6 is the calprotectin light absorption analysis figure of embodiments of the invention 1.Sample calprotectin concentration passes through BCA eggs
White concentration mensuration method obtains（0.791mg/mL）, sample A280 is 0.682, and every mg/mL calprotectins can be obtained according to this result
Corresponding A 280 is about 0.862.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with the accompanying drawings and specific implementation
Example is described in further detail to the present invention.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention,
It is not intended to limit the present invention.
In this manual, " impurity protein " is other albumen in addition to calprotectin.
A kind of separation of the present invention and the method for purifying calprotectin, in accordance with the following steps：
First, the leukocyte samples for including the calprotectin are obtained.By taking people's whole blood as an example, people's whole blood is centrifuged；Separation
Leukocytic cream between red blood cell layer and plasma layer；Separating red corpuscle layer and with the further splitting erythrocyte of hypotonic solution,
Cell suspension after cracking centrifuge and further collects leucocyte.
Secondly, the leukocyte samples are handled through clasmatosis to obtain the endochylema crude extract comprising the calprotectin.
The methods of method of broken leucocyte includes Mechanical Crushing method, multigelation, ultrasonication；It is it is preferred that even using glass cell
Starch device（glass dounce homogenizer）Mechanical Crushing method.For example, in the first preferred embodiment, with equal
Matter device crushes leucocyte and obtains endochylema crude extract, and the pH of plasmosin extract is 6.5~7.0, in plasmosin extract
Tris-HCl concentration is 15~25mmol/L, NaCl concentration is 1.5~2.5mmol/L, MgCl2Concentration is 3.0~4.0 mmol/
L.In second of preferred embodiment, the preparation method of suppressor proteins crude extract includes：Leucocyte is in clasmatosis/cracking
After being crushed in liquid, preferentially centrifugation removes cell fragment at low speeds, and then high speed centrifugation, acquisition suppressor proteins slightly carry
Liquid.Described low-speed centrifugal condition is：Rotating speed is 800~1000g, and high speed centrifugation condition is that rotating speed is 12000~15500g.
Clasmatosis/lysate composition is:20 mmol/L Tris-HCl, pH 6.5~7.0 or pH7.0~7.5, or pH8.0
~8.5；0-10 mmol/L NaCl;0~10 mmol/L MgCl2,0.1~1 mmol/L DTT, 0~5 mmol/L
EDTA, 0-1 mmol/L EGTA。
Then, ammonium sulfate is added into endochylema crude extract and obtains the comprising the calprotectin to remove impurity protein
One refined solution.Ammonium sulfate is added into endochylema crude extract to ammonium sulfate final concentration of 35%（w/v）, that is to say, that.Ammonium sulfate sinks
After the impurity protein of shallow lake, centrifugation removes precipitation, and centrifugal rotational speed is 12000~15500g；Collection supernatant is the first refined solution, then is passed through
The anion exchange chromatography chromatography of subsequent step, collects purifying protein.
Further, before ammonium sulfate precipitation is added, EDTA can be first added into endochylema crude extract with aids precipitation
Impurity protein obtains the endochylema crude extract comprising the calprotectin.After ammonium sulfate precipitation is added, by the first refined solution
Further filtered.
Then, to obtain the albumen comprising the calprotectin through the first refined solution described in dialysis treatment at least once molten
Liquid.Protein solution is placed in elution buffer and dialysed, the pH of elution buffer is 8.0~8.5, Tris- in elution buffer
HCl concentration is 15~25mmol/L, EDTA concentration is 4.0~6.0mmol/L.
Then, the protein solution is handled through ion-exchange chromatography at least once to obtain washing comprising the calprotectin
De- liquid.
Further, the protein solution ion exchange column is subjected to Image processing first, then entered with cleaning buffer solution
Row cleaning, is finally eluted and is collected the eluent for including the calprotectin with elution buffer；Wherein, the cleaning is slow
The pH of fliud flushing is 8.0~8.5, in cleaning buffer solution Tris-HCl concentration be 15~25mmol/L, EDTA concentration be 4.0~
6.0mmol/L；The pH of the elution buffer is 8.0~8.5, and Tris-HCl concentration is 15~25mmol/ in elution buffer
L, EDTA concentration is 4.0~6.0mmol/L.
Finally, also include handling the eluent through sieve chromatography to be wrapped after the ion exchange chromatography step
The step of the second eluent containing calprotectin.Hepes-NaOH concentration is 20 in chromatography buffer used in sieve chromatography
Mmol/L, NaCl concentration are that the concentration that 0.4 mol/L, PMSF concentration is 0.1 mmol/L, DTT is 0.2 mmol/L, pH
For 8.0, the concentration that Hepes-NaOH concentration is 20 mmol/L, NaCl in elution buffer is the dense of 0.4 mol/L, PMSF
The concentration spent for 0.1 mmol/L, DTT is 0.2 mmol/L, pH 8.0.The above method is miscellaneous based on technology removal endochylema of saltouing
The calprotectin enrichment method of albumen, with reference to anion chromatographic and molecular sieve chromatography chromatography, purify the improvement of calprotectin
Technology, it has the advantages that to greatly improve separation yield and sample purity, the integrality for ensureing protein multimers.PMSF is chemistry
The English abbreviation of benzylated substance sulfuryl fluoride, DTT are dithiothreitol (DTT).
Embodiment 1
A kind of method for separating and purifying calprotectin is present embodiments provided, step is as follows：Step 1. from people's whole blood from
The heart separates the leucocyte between red blood cell and blood plasma, and with the further splitting erythrocyte of hypotonic solution, leucocyte is collected by centrifugation.
Step 2. crushes leucocyte with glass cell homogenizer, obtains plasmosin crude extract, and clasmatosis used and plasmosin are taken out
The composition of extract is 20mM Tris-HCl, pH6.5-7.0;2mM NaCl;3.5mM MgCl2.Step 3. is in 850g low speed
Centrifuged 10 minutes on centrifuge, keep rotary head under the conditions of 4 DEG C.Step 4. collects endochylema supernatant, adds EDTA final concentrations
For 5mM, after mixing, centrifuged 30 minutes on 15500g supercentrifuges, keep rotary head temperature at 4 DEG C.Step 5. collect from
The endochylema supernatant of heart separation, ammonium sulfate is added to ammonium sulfate final concentration of 35%（w/v）, mixed under the conditions of 4 DEG C equal
It is even.After step 6. formation to be precipitated, centrifuged 30 minutes on 15500g supercentrifuges, keep rotary head temperature at 4 DEG C.Step
Rapid 7. collect supernatant, filter out the precipitation that may be mixed, are dialysed in 20L elution buffers 2 times, every time 5 hours.Dialysis
Buffer solution forms：20mmol/L Tris-HCl；pH 8.0-8.5;5mmol/L EDTA.Step 8. is by the albumen after dialysis
Solution is added to anion exchange chromatography and is chromatographed and use cleaning buffer solution（20mmol/L Tris-HCl,pH 8.0-
8.5;5mmol/L EDTA）Cleaning, then uses elution buffer（20mmol/L Tris-HCl,pH 8.0-8.5;1mol/L
NaCl;5mmol/L EDTA）Protein sample is eluted, eluent 5mL/ pipes is collected, monitors and elute by ultraviolet specrophotometer A280
The protein concentration of liquid, as a result as shown in Figure 1.
Step 9. draws a small amount of eluent and carries out albuminous degeneration gel electrophoresis (SDS-PAGE), with coomassie brilliant blue staining, choosing
Calprotectin enriched composition, mixing are taken, is chromatographed for further molecular sieve chromatography, as a result as shown in Figure 2.
The eluent of mixing is concentrated into 5mL by step 10., is added to concentrate in molecular sieve chromatography chromatographic column and carries out layer
Analysis.Concentrate can be obtained by ultrafiltration.
Step 11. elution, the calprotectin for obtaining purifying, as a result as shown in Figure 3.
Step 12. draws a small amount of eluent and carries out albuminous degeneration gel electrophoresis electrophoresis（SDS-PAGE）, coomassie brilliant blue staining,
Choose calprotectin enriched composition, mix, the calprotectin purified, as a result as shown in Figure 4.
Step 13. determines A280, and corresponding to 1mg albumen/mL by 0.862 calculates albumen yield, from 1000mL human leukocytes
Endochylema lysate can obtain the calprotectin of 30mg purifying, and the qualification result of calprotectin is as shown in Figure 5 and Figure 6.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (10)
- A kind of 1. method for separating and purifying calprotectin, it is characterised in that this method comprises the following steps：a）Obtain the leukocyte samples for including the calprotectin；b）The leukocyte samples are handled through clasmatosis to obtain the endochylema crude extract comprising the calprotectin；c）Ammonium sulfate is added into endochylema crude extract and obtains the first purifying comprising the calprotectin to remove impurity protein Liquid；d）The protein solution for including the calprotectin is obtained through the first refined solution described in dialysis treatment at least once；e）The protein solution is handled through ion-exchange chromatography at least once to obtain the eluent for including the calprotectin；Withf）The eluent is handled through sieve chromatography to obtain the second eluent comprising calprotectin.
- 2. the method according to claim 1 for separating and purifying calprotectin, it is characterised in that in step b）Afterwards, walk Rapid c）Also comprise the following steps before：It is thick to obtain the endochylema comprising the calprotectin with aids precipitation impurity protein that EDTA is added into endochylema crude extract Extract.
- 3. the method according to claim 1 for separating and purifying calprotectin, it is characterised in that in step d）In, dialysis The pH of buffer solution is 8.0~8.5, in elution buffer Tris-HCl concentration be 15~25mmol/L, EDTA concentration be 4.0~ 6.0mmol/L。
- 4. the method according to claim 1 for separating and purifying calprotectin, it is characterised in that in step e）In, first The protein solution is subjected to Image processing by ion exchange column, then cleaned with cleaning buffer solution, finally with elution Buffer solution is eluted and collected the eluent for including the calprotectin；Wherein, the pH of the cleaning buffer solution be 8.0~ 8.5, in cleaning buffer solution Tris-HCl concentration be 15~25mmol/L, EDTA concentration be 4.0~6.0mmol/L；The elution The pH of buffer solution is 8.0~8.5, in elution buffer Tris-HCl concentration be 15~25mmol/L, EDTA concentration be 4.0~ 6.0mmol/L。
- 5. the method according to claim 1 for separating and purifying calprotectin, it is characterised in that ammonium sulfate concentrations 35% w/v。
- 6. the method according to claim 1 for separating and purifying calprotectin, it is characterised in that the step a）Specially The leukocyte samples for including the calprotectin are obtained from people's whole blood.
- 7. the method according to claim 1 for separating and purifying calprotectin, it is characterised in that first refined solution enters One step carries out filtration step.
- 8. the method according to claim 1 for separating and purifying calprotectin, it is characterised in that in step b）In, with equal Matter device crushes leucocyte and obtains endochylema crude extract, and the pH of plasmosin extract is 6.5~7.0, in plasmosin extract Tris-HCl concentration is 15~25mmol/L, NaCl concentration is 1.5~2.5mmol/L, MgCl2Concentration is 3.0~4.0 mmol/ L。
- 9. a kind of separate and purify the kit of calprotectin, it is characterised in that the kit includes：For extracting the plasmosin extract of cell protein；For removing the ammonium sulfate of impurity protein；Elution buffer for dialysis treatment；For cleaning the cleaning buffer solution of chromatographic column；Elution buffer for elution chromatography post；WithFor eluting the second elution buffer of molecular sieve.
- 10. according to claim 9 separate and purify the kit of calprotectin, it is characterised in that the kit also wraps Include：For removing deimpurity EDTA solution.
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| JPS62116519A (en) * | 1985-07-18 | 1987-05-28 | Ichimaru Fuarukosu Kk | Cell metabolism activating substance extracted from placenta, extraction thereof and method for inactivating hepatitis virus |
| CN1800202A (en) * | 2006-01-20 | 2006-07-12 | 南京师范大学 | Method for extracting purified high ferro myoglobins from cardiac muscle |
| CN105181977A (en) * | 2015-11-05 | 2015-12-23 | 四川沃文特生物技术有限公司 | Kit for detecting calprotectin in excrement |
| CN206038696U (en) * | 2016-03-09 | 2017-03-22 | 广州沣润生物科技有限公司 | Ration calprotectin detects immunochromatographic test strip |
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2017
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|---|---|---|---|---|
| JPS62116519A (en) * | 1985-07-18 | 1987-05-28 | Ichimaru Fuarukosu Kk | Cell metabolism activating substance extracted from placenta, extraction thereof and method for inactivating hepatitis virus |
| CN1800202A (en) * | 2006-01-20 | 2006-07-12 | 南京师范大学 | Method for extracting purified high ferro myoglobins from cardiac muscle |
| CN105181977A (en) * | 2015-11-05 | 2015-12-23 | 四川沃文特生物技术有限公司 | Kit for detecting calprotectin in excrement |
| CN206038696U (en) * | 2016-03-09 | 2017-03-22 | 广州沣润生物科技有限公司 | Ration calprotectin detects immunochromatographic test strip |
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