CN110339209A - A kind of preparation method for the calf serum going Thyroid binding protein - Google Patents
A kind of preparation method for the calf serum going Thyroid binding protein Download PDFInfo
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- CN110339209A CN110339209A CN201910650816.1A CN201910650816A CN110339209A CN 110339209 A CN110339209 A CN 110339209A CN 201910650816 A CN201910650816 A CN 201910650816A CN 110339209 A CN110339209 A CN 110339209A
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- 210000002966 serum Anatomy 0.000 title claims abstract description 124
- 244000309466 calf Species 0.000 title claims abstract description 104
- 210000001685 thyroid gland Anatomy 0.000 title claims abstract description 36
- 108091008324 binding proteins Proteins 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 102000014914 Carrier Proteins Human genes 0.000 title claims abstract 5
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 24
- 238000010521 absorption reaction Methods 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 16
- 238000001179 sorption measurement Methods 0.000 claims abstract description 16
- 238000001914 filtration Methods 0.000 claims description 13
- 239000012528 membrane Substances 0.000 claims description 9
- 230000001681 protective effect Effects 0.000 claims description 8
- 238000012856 packing Methods 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 5
- 239000008280 blood Substances 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 2
- 102000002322 Egg Proteins Human genes 0.000 claims description 2
- 108010000912 Egg Proteins Proteins 0.000 claims description 2
- 150000001450 anions Chemical class 0.000 claims description 2
- 235000014103 egg white Nutrition 0.000 claims description 2
- 210000000969 egg white Anatomy 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims 1
- 239000012895 dilution Substances 0.000 claims 1
- 239000011347 resin Substances 0.000 claims 1
- 229920005989 resin Polymers 0.000 claims 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 abstract description 9
- 229940034208 thyroxine Drugs 0.000 abstract description 9
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 238000005259 measurement Methods 0.000 abstract description 3
- 102000023732 binding proteins Human genes 0.000 description 29
- 230000000052 comparative effect Effects 0.000 description 10
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 241000283690 Bos taurus Species 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 238000003257 protein preparation method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 101800000263 Acidic protein Proteins 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102100025292 Stress-induced-phosphoprotein 1 Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000005414 inactive ingredient Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000012609 strong anion exchange resin Substances 0.000 description 1
- 229940035722 triiodothyronine Drugs 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Immunology (AREA)
- Virology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of preparation methods of calf serum for going Thyroid binding protein, the described method comprises the following steps: (1) adsorbing calf serum using anion exchange resin, collect the calf serum of outflow；(2) calf serum by step (1) after absorption carries out chromatographic adsorption, the calf serum after obtaining second of absorption；(3) calf serum obtained in step (2) after second adsorbs is subjected to ultrafiltration, obtains the calf serum of Thyroid binding protein；The present invention can be realized thyroxine T3 in calf serum, the basic removal of T4, not interference measurement, TT3 content is 0.85pmol/L~0.96pmol/L in finally obtained calf serum, TT4 content is 1.33pmol/L~1.44pmol/L, and production cost is acceptable, it can be achieved that industrialization.
Description
Technical field
The invention belongs to bio-separation fields, are related to a kind of preparation method of calf serum for going Thyroid binding protein.
Background technique
The existing way of thyroxine T3 in calf serum, T4 in serum is there are mainly two types of form, one is free,
One is the form presence to combine with Thyroid binding protein.The T3 to dissociate in serum, T4 serum can be gone with active carbon adsorption
It removes, but in serum and thyroxine T3 that Thyroid binding protein combines, T4 are difficult to remove.Contain several hundred kinds mainly in serum
Albumen, some specific protein removal difficulty is big, and cost is also very high.
CN100531723A discloses calf serum protein-removing extract for injection and preparation method thereof, which goes
Protein extract freezing-dried powder injection, the component containing following portions by weight ratio: calf serum protein-removing extract dry matter 1
Part；9-11 parts of mannitol；4.5-5.5 parts of small molecule dextran.Calf serum protein-removing extract freeze-dried powder of the invention
Injection overcomes water needle and the unstable defect of injection quality, extends keeping life, extends clinical application, reduces bad anti-
Answer odds.
CN100473387A discloses a kind of calf serum de-protein injection and its production method, the injection be according to
Following method preparation: 1) removing the albumen in calf serum, then carry out coarse filtration, collect filtrate；2) successively to the filtrate
It is extracted with toluene, ethyl acetate and n-butanol, discards organic phase in extraction every time and retain water phase；3) water phase is used
BIO-GEL-P2 gel is chromatographed, and calf serum de-protein injection is obtained.The work of the calf serum de-protein injection
Property ingredient be polysaccharide, the content of the polysaccharide is 1.0-1.2mg/mL, preferably 1.17mg/mL.This method only uses gel layer
Analysis can not remove albumen thorough.
CN102805754A discloses a kind of purification process of calf serum protein-removing extract, this method comprises: adopting children
Ox venous blood, isolates serum, and with ethyl alcohol deproteinized, decompression removal ethyl alcohol is added composite protease hydrolysis, is centrifuged after hydrolysis
Supernatant, supernatant ultrafiltration are stayed in separation；For ultrafiltrate first with glucan G-15 gel chromatography desalination, collecting at 280nm has specificabsorption
Elution fraction；With the separation of DEAE-Sephadex-50 gel chromatography, the elution portion for having specificabsorption at second 280nm is collected
Point；It is virus inactivated using micro-pore-film filtration and ultraviolet light irradiation；Obtain calf serum protein-removing extract.This method is only
Combined using the step of gel chromatography and ultrafiltration, although removing many inactive ingredients, reduce product molecular weight ranges and
The probability that adverse reaction occurs, improves purity, safety and the bioactivity of product, but the effect removed still have it is to be added
By force.
Therefore, a kind of method for efficiently removing thyroxine T3, T4 in calf serum how is developed, for calf serum
Using having great importance.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of calf serums for going Thyroid binding protein
Preparation method, with achieve the purpose that high efficiency remove Thyroid binding protein.
To achieve this purpose, the present invention adopts the following technical scheme:
The present invention provides a kind of preparation methods of calf serum for going Thyroid binding protein, and the method includes following
Step:
(1) calf serum is adsorbed using anion exchange resin, collects the calf serum of outflow；
(2) calf serum by step (1) after absorption carries out chromatographic adsorption, the small ox blood after obtaining second of absorption
Clearly；
(3) calf serum obtained in step (2) after second adsorbs is subjected to ultrafiltration, obtains thyroid gland knot
The calf serum of hop protein.
The preparation method of the calf serum provided by the invention for going Thyroid binding protein, using the method for ion exchange,
Most of Thyroid binding protein is first removed, affinity chromatography is added, Thyroid binding protein is almost removed, is realized small
The basic removal of thyroxine T3 in cow's serum, T4, interference measurement, production cost be not acceptable, it can be achieved that industrialization.
In the present invention, the Thyroid binding protein in calf serum is acidic protein, isoelectric pH value 5.1, in yin
It can be adsorbed on ion exchange resin, the primary protein component in serum, such as albumin, in defined conditions, substantially not
Absorption.By the chromatography of step (1) in removal serum most of Thyroid binding protein, reach T3 in removal serum, T4's
Purpose, while remaining the primary protein component in serum.The absorption of strong anion exchange resin can remove 95% first shape
Gland binding protein realizes that T3, T4 are removed substantially.
In the present invention, the dilute serum adsorbed by anion exchange resin will be remaining by further chromatographic adsorption
5% bovine thyroid binding protein almost remove completely.Chromatography cost in step (2) is a highest step in technique.But
Adsorption effect is good, can be Reusability 50 times after regeneration, says from industrialization angle, and the cost for realizing product preparation is controllable.
For the two step chromatographic adsorptions for going on smoothly front, starting is used for the calf serum of Anion-adsorption, is first diluted,
It is generated with reducing protein concentration and polymer, guarantees the chromatography behavior of albumen not by the shadow of the viscosity of albumen and intermolecular combination
It rings.Dilute serum after two step chromatographic adsorptions, protein concentration are lower than several times of protein concentration of serum, further use the side of ultrafiltration
The protein concentration of serum after absorption is reached or approached the concentration of normal calf serum, aseptic filtration, packing, Cheng Wujia by method
The serum product of shape parathyrine T3, T4.Find thyroxine T3, T4 for dissociating in serum in first step anion in early-stage study
Also be removed simultaneously in adsorption process, be since the hydrophobic framework of anion exchange resin may be by hydrophobic effect, will be hydrophobic
Property thyroxine T3, T4 absorption fall.
In general, the TT3 mean value of calf serum is 2.3nmol/L, and TT4 is the close of 90nmol/L and people, free
Mean value is that FT3 is 20pmol/L, FT4 1.07nmol/L.And method of the invention is used, being finally reached can make TT3 contain
Amount is in 1pmol/L hereinafter, the content of TT4 is in 1.5pmol/L or less.
Wherein, TT3 is total triiodo thryonine, and TT4 is total thyroxin, and FT3 is free triiodothyronine,
FT4 is free thyroxine.
In the present invention, calf serum is generally diluted using water.
Preferably, before being adsorbed described in step (1), calf serum is diluted.
Preferably, the diluted extension rate is 2~6 times, such as can be 2 times, 3 times, 4 times, 5 times or 6 times etc., excellent
It is selected as 4~5 times.
Preferably, the model D201 macroporous anion exchange resin of anion exchange resin described in step (1).
Preferably, flow velocity of the calf serum in anion exchange resin is 5~30mL/min, such as be can be
5mL/min、6mL/min、7mL/min、8mL/min、10mL/min、12mL/min、15mL/min、20mL/min、25mL/min、
28mL/min or 30mL/min etc..
Preferably, chromatography described in step (2) is to be chromatographed using DEAE-Sephadex-50.
Preferably, the dosage of the DEAE-Sephadex-50 is 0.02~0.5 times of calf serum quality, such as can be with
It is 0.02 times, 0.05 times, 0.08 times, 0.1 times, 0.2 times, 0.3 times, 0.4 times or 0.5 times etc..
In the present invention, good chromatographic effect can be realized using less dosage.
Preferably, the aperture of ultrafiltration membrane described in step (3) is 0.5 μm~5 μm, such as can be 0.5 μm, 0.8 μm, 1 μ
M, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm or 5 μm etc..
Preferably, further include the steps that aseptic filtration after ultrafiltration described in step (3).
Preferably, further include that the calf serum that will be obtained dispenses after aseptic filtration described in step (3), then-
The step of 20 DEG C of freezen protectives.
Preferably, the preparation method comprises the following steps:
(1) calf serum is diluted 2~6 times, is adsorbed using D201 macroporous anion exchange resin, controlled small
The flow velocity of cow's serum is 5~30mL/min, collects the calf serum of outflow；
(2) calf serum by step (1) after absorption carries out chromatographic adsorption using DEAE-Sephadex-50,
The dosage of DEAE-Sephadex-50 is 0.02~0.5 times of calf serum quality, the calf serum after obtaining second of absorption；
It (3) the use of aperture is 0.5 μm~5 μm by the calf serum obtained in step (2) after second adsorbs
Ultrafiltration membrane carry out ultrafiltration, then aseptic filtration, after packing at -20 DEG C freezen protective, obtain the small of Thyroid binding protein
Cow's serum.
The present invention will bovine thyroid binding protein after purification, as antigen, immune mouse is prepared for bovine thyroid combination egg
White monoclonal antibody prepares ascites, antibody purification with mouse, then is coupled with Ago-Gel.
Compared with the existing technology, the invention has the following advantages:
The preparation method of the calf serum provided by the invention for going Thyroid binding protein, using the method for ion exchange,
Most of Thyroid binding protein is first removed, affinity chromatography is added, Thyroid binding protein is almost removed, is realized small
The basic removal of thyroxine T3 in cow's serum, T4, not interference measurement, TT3 content is in finally obtained calf serum
0.85pmol/L~0.96pmol/L, TT4 content are 1.33pmol/L~1.44pmol/L, production cost it is acceptable, it can be achieved that
Industrialization.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1
The present embodiment provides a kind of preparation methods of calf serum for going Thyroid binding protein
(1) calf serum is diluted 3 times, is adsorbed using D201 macroporous anion exchange resin, control calf
The flow velocity of serum is 25mL/min, collects the calf serum of outflow；
(2) calf serum by step (1) after absorption carries out chromatographic adsorption using DEAE-Sephadex-50,
The dosage of DEAE-Sephadex-50 is 0.05 times of calf serum quality, the calf serum after obtaining second of absorption；
(3) by the calf serum obtained in step (2) after second adsorbs using aperture be 3 μm ultrafiltration membrane into
Row ultrafiltration, then aseptic filtration, after packing at -20 DEG C freezen protective, obtain the calf serum of Thyroid binding protein.
Embodiment 2
The present embodiment provides a kind of preparation methods of calf serum for going Thyroid binding protein
(1) calf serum is diluted 2 times, is adsorbed using D201 macroporous anion exchange resin, control calf
The flow velocity of serum is 5mL/min, collects the calf serum of outflow；
(2) calf serum by step (1) after absorption carries out chromatographic adsorption using DEAE-Sephadex-50,
The dosage of DEAE-Sephadex-50 is 0.02 times of calf serum quality, the calf serum after obtaining second of absorption；
(3) ultrafiltration membrane for the use of aperture being 0.5 μm by the calf serum obtained in step (2) after second adsorbs
Carry out ultrafiltration, then aseptic filtration, after packing at -20 DEG C freezen protective, obtain the calf serum of Thyroid binding protein.
Embodiment 3
The present embodiment provides a kind of preparation methods of calf serum for going Thyroid binding protein
(1) calf serum is diluted 6 times, is adsorbed using D201 macroporous anion exchange resin, control calf
The flow velocity of serum is 30mL/min, collects the calf serum of outflow；
(2) calf serum by step (1) after absorption carries out chromatographic adsorption using DEAE-Sephadex-50,
The dosage of DEAE-Sephadex-50 is 0.5 times of calf serum quality, the calf serum after obtaining second of absorption；
(3) by the calf serum obtained in step (2) after second adsorbs using aperture be 5 μm ultrafiltration membrane into
Row ultrafiltration, then aseptic filtration, after packing at -20 DEG C freezen protective, obtain the calf serum of Thyroid binding protein.
Embodiment 4
The present embodiment provides a kind of preparation methods of calf serum for going Thyroid binding protein
(1) calf serum is diluted 4 times, is adsorbed using D201 macroporous anion exchange resin, control calf
The flow velocity of serum is 20mL/min, collects the calf serum of outflow；
(2) calf serum by step (1) after absorption carries out chromatographic adsorption using DEAE-Sephadex-50,
The dosage of DEAE-Sephadex-50 is 0.04 times of calf serum quality, the calf serum after obtaining second of absorption；
(3) by the calf serum obtained in step (2) after second adsorbs using aperture be 4 μm ultrafiltration membrane into
Row ultrafiltration, then aseptic filtration, after packing at -20 DEG C freezen protective, obtain the calf serum of Thyroid binding protein.
Embodiment 5
The present embodiment the difference from embodiment 1 is that, flow velocity in the present embodiment in step (1) is 2mL/min.
Embodiment 6
The present embodiment the difference from embodiment 1 is that, flow velocity in the present embodiment in step (1) is 35mL/min.
Embodiment 7
The present embodiment the difference from embodiment 1 is that, in the present embodiment in step (3) ultrafiltration membrane aperture be 0.1 μm.
Embodiment 8
The present embodiment the difference from embodiment 1 is that, in the present embodiment in step (3) ultrafiltration membrane aperture be 6 μm.
Comparative example 1
This comparative example the difference from embodiment 1 is that, in this comparative example include step (1).
Comparative example 2
This comparative example the difference from embodiment 1 is that, in this comparative example include step (2).
Comparative example 3
This comparative example the difference from embodiment 1 is that, in this comparative example include step (3).
Calf serum in embodiment 1-8 and comparative example 1-3 is subjected to TT3 and TT4 content detection, as a result such as the following table 1 institute
Show:
Table 1
Data in consolidated statement 1 it is found that the calf serum provided by the invention for going Thyroid binding protein preparation method
Removal effect is very good, and the TT3 content finally measured is in 0.85pmol/L~0.96pmol/L, and TT4 content is in 1.33pmol/L
~1.44pmol/L, content are very low.
And if it will affect the effect of removal after flow velocity in step (1), the filter sizes in step (3) change；Such as
Fruit lacks any one step in 3 steps, and removal effect declines to a great extent, and is unfavorable for actual production and batch is supplied.
The Applicant declares that the present invention is explained by the above embodiments the of the invention small ox blood for going Thyroid binding protein
Clear preparation method, but the invention is not limited to above-mentioned method detaileds, that is, it is above-mentioned detailed not mean that the present invention must rely on
Method could be implemented.It should be clear to those skilled in the art, any improvement in the present invention, each to product of the present invention
The equivalence replacement of raw material and addition, the selection of concrete mode of auxiliary element etc. all fall within protection scope of the present invention and openly
Within the scope of.
Claims (10)
1. a kind of preparation method for the calf serum for going Thyroid binding protein, which is characterized in that the method includes following steps
It is rapid:
(1) calf serum is adsorbed using anion exchange resin, collects the calf serum of outflow；
(2) calf serum by step (1) after absorption carries out chromatographic adsorption, the calf serum after obtaining second of absorption；
(3) calf serum obtained in step (2) after second adsorbs is subjected to ultrafiltration, obtains thyroid gland combination egg
White calf serum.
2. preparation method according to claim 1, which is characterized in that described in step (1) adsorb before, by calf serum into
Row dilution；
Preferably, the diluted extension rate is 2~6 times；Preferably 4~5 times.
3. preparation method according to claim 1 or 2, which is characterized in that anion exchange resin described in step (1)
Model D201 macroporous anion exchange resin.
4. preparation method according to any one of claim 1-3, which is characterized in that the calf serum is handed in anion
The flow velocity changed in resin is 5~30mL/min.
5. preparation method described in any one of -4 according to claim 1, which is characterized in that chromatography described in step (2) is to make
It is chromatographed with DEAE-Sephadex-50.
6. preparation method according to any one of claims 1-5, which is characterized in that the DEAE-Sephadex-50's
Dosage is 0.02~0.5 times of calf serum quality.
7. preparation method according to claim 1 to 6, which is characterized in that ultrafiltration membrane described in step (3)
Aperture is 0.5 μm~5 μm.
8. preparation method described in any one of -7 according to claim 1, which is characterized in that after ultrafiltration described in step (3) also
Include the steps that aseptic filtration.
9. preparation method according to claim 1 to 8, which is characterized in that aseptic filtration described in step (3)
It afterwards further include that the calf serum that will be obtained dispenses, then -20 DEG C of freezen protectives the step of.
10. preparation method according to claim 1 to 9, which is characterized in that the preparation method includes following
Step:
(1) calf serum is diluted 2~6 times, is adsorbed using D201 macroporous anion exchange resin, control small ox blood
Clear flow velocity is 5~30mL/min, collects the calf serum of outflow；
(2) calf serum by step (1) after absorption carries out chromatographic adsorption, DEAE- using DEAE-Sephadex-50
The dosage of Sephadex-50 is 0.02~0.5 times of calf serum quality, the calf serum after obtaining second of absorption；
(3) ultrafiltration for the use of aperture being 0.5 μm~5 μm by the calf serum obtained in step (2) after second adsorbs
Film carry out ultrafiltration, then aseptic filtration, after packing at -20 DEG C freezen protective, obtain the small ox blood of Thyroid binding protein
Clearly.
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Denomination of invention: Preparation method of bovine serum for removing thyroid binding protein Granted publication date: 20230606 Pledgee: Guotou Taikang Trust Co.,Ltd. Pledgor: SHANGHAI TITAN TECHNOLOGY Co.,Ltd. Registration number: Y2024310001016 |