CN104450656A - Method for preparing and purifying thrombin in rabbit blood - Google Patents

Method for preparing and purifying thrombin in rabbit blood Download PDF

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Publication number
CN104450656A
CN104450656A CN201410454654.1A CN201410454654A CN104450656A CN 104450656 A CN104450656 A CN 104450656A CN 201410454654 A CN201410454654 A CN 201410454654A CN 104450656 A CN104450656 A CN 104450656A
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Prior art keywords
zymoplasm
rabbit
centrifugal
loading
solution
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刘晓明
葛安山
荆荣燕
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QINGDAO ZHONGCHUANG BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
KANGDA FOOD CO Ltd QINGDAO
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QINGDAO ZHONGCHUANG BIOLOGICAL SCIENCE & TECHNOLOGY Co Ltd
KANGDA FOOD CO Ltd QINGDAO
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Priority to CN201410454654.1A priority Critical patent/CN104450656A/en
Publication of CN104450656A publication Critical patent/CN104450656A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention discloses a method for preparing and purifying thrombin in rabbit blood. The method mainly comprises the following process steps: (1) pretreating rabbit blood; (2) desalting a filtrate; (3) activating rabbit thrombin; (4) carrying out Heparin affinity chromatography; and (5) purifying rabbit thrombin. The method is simple in operation, the extracted thrombin has high purity, the purity of thrombin in a thrombin stock liquid is 99% and the extraction amount is increased by 15% in comparison with that of the existing method.

Description

The method for preparing purified of zymoplasm in rabbit blood
Technical field
The invention belongs to bioprotein technical field, be specifically related to the method for preparing purified of zymoplasm in rabbit blood.
Background technology
Rabbit blood contains very high nutritive value, can be processed into multiple product, edible, medicinal, or as the animal feedstuff of livestock and poultry.Rabbit blood can extract multiple bio-pharmaceutical and biochemical reagents, as medical serum, serum antigen, zymoplasm, leucine, peptone etc., rabbit blood medical value is high, but rabbit blood extractive technique is only limitted to laboratory applications at present, purification cannot be reached, and industrialization can not extract and produce, the application of rabbit blood is had some limitations.Zymoplasm is mainly used in the production etc. of clinical hemostasis, clotting reagent, but be mainly derived from ox blood at present, the present invention mainly focuses on the extraction purification in enormous quantities of rabbit blood clotting hemase, present method has abandoned the acetone treatment method in traditional method, according to molecular weight and the iso-electric point of zymoplasm, utilize affinity chromatography, improve purity and the output of zymoplasm.
Summary of the invention
In order to overcome the technological deficiency that prior art exists, the invention provides the method for preparing purified of zymoplasm in a kind of rabbit blood, the method is simple to operate, and the zymoplasm purity of extraction is high.
The method for preparing purified of zymoplasm in rabbit blood, mainly comprises following processing step:
(1) rabbit blood pre-treatment
A. fresh for 50L rabbit blood is added in Sodium Citrate (Trisodium Citrate) solution of 4.0% in the ratio of 16:1, and stir evenly;
B. continuous flow centrifuge is centrifugal, and centrifugal force is about 400g, and centrifugal supernatant is blood plasma, and throw out is the cells such as blood cell, collects supernatant;
C. the supernatant collected is passed through depth filter, collect filtrate;
(2) filtrate desalination
Adopt G25 desalination chromatography, by the filtrate of 20L by loading pipeline loading, water influent pipeline is switched to after completion of the sample, when UV-detector detects that albumen flows out, switch to collecting, until UV-detector signal value is 1/10 of its peak value, switch to waste liquid, until UV-detector signal is flat, repeat loading;
(3) activation of rabbit zymoplasm
A. cryoprecipitate: spent the night by the environment that the collection liquid of G25 desalting column is placed in 4 DEG C, it can separate out a large amount of throw outs, this throw out is the mixture of rabbit thrombogen and other protein;
B. centrifugal: by the solution of cryoprecipitate under the condition of 4 DEG C, centrifugal about 30min, collecting precipitation;
C. filter: precipitation 5mMHAc-NaAc solution is cleaned 2 times, then with the PB solubilize that 10mM pH is 7.0, then by the filter paper filtering of 0.45um, obtains prothrombin solution;
D. activate: be 10%CaCl by massfraction in advance 2solution and prothrombin solution are placed in the water-bath of 37 DEG C, continue to add CaCl after 5min in prothrombin solution 2solution, is 1% to its concentration, and mixes at once, terminates to activate, and be transferred to rapidly 4 DEG C after 15min;
(4) Heparin affinity chromatography
The rabbit zymoplasm activated in step (3) is passed through loading pipeline loading, adopts chromatography column to carry out Image processing by Heparin affinity chromatography.
(5) purifying of rabbit zymoplasm
A. ultrafiltration: the molecular weight cut-off of ultra-fine filter is 10KD, and membrane area is 200cm 2;
Fluid inlet is put into PBS damping fluid, before its volume is about 1/10 time, by water influent pipeline and return line PBS damping fluid collect.
B. centrifugal: by the zymoplasm elutriant after ultrafiltration under the condition of 4 DEG C, centrifugal about 30min, collects supernatant, is zymoplasm stoste.
Described G25 chromatography column is chromatography column 450*600, and filler is G25, and loading height is 50cm, about needs the G25 filler of 80L.
Described Heparin affinity chromatography adopts chromatography column 100*300, and filler is Heparin 6FF, and loading height is 10cm, about needs 0.6L filler, and described Buffer A is 10mM PB, pH 7.0; Described Buffer B is Buffer A+250mM NaCl.
Be 99% by zymoplasm purity in present invention process step gained zymoplasm stoste, extracted amount improves 15% than existing method.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
The method for preparing purified of zymoplasm in rabbit blood, mainly comprises following processing step:
(1) rabbit blood pre-treatment
A. fresh for 50L rabbit blood is added in Sodium Citrate (Trisodium Citrate) solution of 4.0% in the ratio of 16:1, and stir evenly.
B. continuous flow centrifuge is centrifugal, and centrifugal force is about 400g, and centrifugal supernatant is blood plasma, and throw out is the cells such as blood cell, collects supernatant.
C. the supernatant collected is passed through depth filter, collect filtrate.
(2) filtrate desalination
Adopt G25 desalination chromatography, chromatography column 450*600, filler is G25, and loading height is 50cm, about needs the G25 filler of 80L, and Buffer:5mM HAC-NaAC, pH are 5.2.
By the filtrate of 20L by loading pipeline loading, water influent pipeline 1 is switched to after completion of the sample, when UV-detector detects that albumen flows out, switch to collecting 1, until UV-detector signal value is 1/10 of its peak value, switch to waste liquid, until UV-detector signal is flat, repeat loading.
Buffer is extracted by water influent pipeline, and balance chromatography column, until pH is 5.2, and UV-detector signal is flat.1M NaOH is extracted by water influent pipeline 2, carries out CIP(incumbent firms to chromatography column), until pH is 13 or more and UV-detector signal is flat, and keep 30min.
DdH2O is extracted by water influent pipeline 3, rinses chromatography column, until its pH value is 7 or following and UV-detector signal is flat.
20% ethanol is extracted by water influent pipeline 4, rinses chromatography column, about 1.5 column volumes.
(3) activation of rabbit zymoplasm
A. cryoprecipitate: spent the night by the environment that the collection liquid of G25 desalting column is placed in 4 DEG C, it can separate out a large amount of throw outs, this throw out is the mixture of rabbit thrombogen and other protein;
C. centrifugal: by the solution of cryoprecipitate under the condition of 4 DEG C, centrifugal about 30min, collecting precipitation;
C. filter: precipitation 5mMHAc-NaAc solution is cleaned 2 times, then with the PB solubilize that 10mM pH is 7.0, then by the filter paper filtering of 0.45um, obtains prothrombin solution;
D. activate: be 10%CaCl by massfraction in advance 2solution and prothrombin solution are placed in the water-bath of 37 DEG C, continue to add CaCl after 5min in prothrombin solution 2solution, is 1% to its concentration, and mixes at once, terminates to activate, and be transferred to rapidly 4 DEG C after 15min;
(4) Heparin affinity chromatography
The rabbit zymoplasm activated in step (3) is passed through loading pipeline loading, adopts chromatography column to carry out Image processing by Heparin affinity chromatography.
Adopt chromatography column 100*300, filler is Heparin 6FF, and loading height is 10cm, about needs 0.6L filler, and described Buffer A is 10mM PB, pH 7.0; Described Buffer B is Buffer A+250mM NaCl.
Extracted by water influent pipeline 1 by Buffer A, balance chromatography column, until pH is 7.0, and UV-detector signal is flat.By all activated zymoplasm by loading pipeline loading, after completion of the sample, be switched to water influent pipeline 1.Continuation Buffer A rinses chromatography column until UV-detector signal is flat.Buffer B is extracted by water influent pipeline 2, when UV-detector detects that albumen flows out, switches to collecting 1, until UV-detector signal value is 1/10 of its peak value, switch to waste liquid, until UV-detector signal is flat.1M NaOH is extracted by water influent pipeline 3, carries out CIP(incumbent firms to chromatography column), until pH is 13 or more and UV-detector signal is flat.Buffer B is extracted by water influent pipeline 2, rinses chromatography column, until its pH value is 7 or following and UV-detector signal is flat.20% ethanol is extracted by water influent pipeline 4, rinses chromatography column, about 1.5 column volumes.
(5) purifying of rabbit zymoplasm
A. ultrafiltration: the molecular weight cut-off of ultra-fine filter is 10KD, and membrane area is 200cm 2;
Fluid inlet is put into PBS damping fluid, before its volume is about 1/10 time, by water influent pipeline and return line PBS damping fluid collect.
B. centrifugal: by the zymoplasm elutriant after ultrafiltration under the condition of 4 DEG C, centrifugal about 30min, collects supernatant, is zymoplasm stoste.
In addition, the present invention is not limited to above-mentioned embodiment, based on the technique effect that same approach reaches, all belongs to protection scope of the present invention.

Claims (3)

1. the method for preparing purified of zymoplasm in rabbit blood, is characterized in that: mainly comprise following processing step:
(1) rabbit blood pre-treatment
A. fresh for 50L rabbit blood is added in Sodium Citrate (Trisodium Citrate) solution of 4.0% in the ratio of 16:1, and stir evenly;
B. continuous flow centrifuge is centrifugal, and centrifugal force is about 400g, and centrifugal supernatant is blood plasma, and throw out is the cells such as blood cell, collects supernatant;
C. the supernatant collected is passed through depth filter, collect filtrate;
(2) filtrate desalination
Adopt G25 desalination chromatography, by the filtrate of 20L by loading pipeline loading, water influent pipeline is switched to after completion of the sample, when UV-detector detects that albumen flows out, switch to collecting, until UV-detector signal value is 1/10 of its peak value, switch to waste liquid, until UV-detector signal is flat, repeat loading;
(3) activation of rabbit zymoplasm
A. cryoprecipitate: spent the night by the environment that the collection liquid of G25 desalting column is placed in 4 DEG C, it can separate out a large amount of throw outs, this throw out is the mixture of rabbit thrombogen and other protein;
Centrifugal: by the solution of cryoprecipitate under the condition of 4 DEG C, centrifugal about 30min, collecting precipitation;
C. filter: precipitation 5mMHAc-NaAc solution is cleaned 2 times, then with the PB solubilize that 10mM pH is 7.0, then by the filter paper filtering of 0.45um, obtains prothrombin solution;
D. activate: be 10%CaCl by massfraction in advance 2solution and prothrombin solution are placed in the water-bath of 37 DEG C, continue to add CaCl after 5min in prothrombin solution 2, be 1% to its concentration, and mix at once, terminate after 15min to activate, and be transferred to rapidly 4 DEG C;
(4) Heparin affinity chromatography
The rabbit zymoplasm activated in step (3) is passed through loading pipeline loading, adopts chromatography column to carry out Image processing by Heparin affinity chromatography;
(5) purifying of rabbit zymoplasm
A. ultrafiltration: the molecular weight cut-off of ultra-fine filter is 10KD, and membrane area is 200cm 2;
Fluid inlet is put into PBS damping fluid, before its volume is about 1/10 time, by water influent pipeline and return line PBS damping fluid collect;
B. centrifugal: by the zymoplasm elutriant after ultrafiltration under the condition of 4 DEG C, centrifugal about 30min, collects supernatant, is zymoplasm stoste.
2. the method for preparing purified of zymoplasm in rabbit blood according to claim 1, it is characterized in that: described G25 chromatography column is chromatography column 450*600, filler is G25, and loading height is 50cm, about needs the G25 filler of 80L.
3. the method for preparing purified of zymoplasm in rabbit blood according to claim 1, is characterized in that: described Heparin affinity chromatography adopts chromatography column 100*300, and filler is Heparin 6FF, loading height is 10cm, about need 0.6L filler, described Buffer A is 10mM PB, pH 7.0; Described Buffer B is Buffer A+250mM NaCl.
CN201410454654.1A 2014-09-09 2014-09-09 Method for preparing and purifying thrombin in rabbit blood Pending CN104450656A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105039273A (en) * 2015-07-13 2015-11-11 青岛康大食品有限公司 Method for purifying and preparing superoxide dismutase in rabbit blood
CN105039295A (en) * 2015-09-15 2015-11-11 上海洲跃生物科技有限公司 Method for preparing human thrombin from cold-removing glue plasma
CN108473530A (en) * 2015-10-15 2018-08-31 普莱泽玛科技有限公司 The method for extracting proteins from blood plasma
CN108660126A (en) * 2018-06-08 2018-10-16 博雅生物制药集团股份有限公司 A kind of preparation process of freeze dried human zymoplasm

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1587401A (en) * 2004-09-15 2005-03-02 黄耀江 Process for preparing thrombase
CN101067132A (en) * 2006-11-17 2007-11-07 马忠仁 Thrombin extracting and separating process
CN101306353A (en) * 2008-02-02 2008-11-19 中国人民解放军南京军区南京总医院 Heparin affinity column and preparation method and use thereof
CN101979532A (en) * 2010-10-13 2011-02-23 江苏省江大绿康生物工程技术研究有限公司 Method for comprehensively using pig blood
CN203763551U (en) * 2013-12-26 2014-08-13 中原大学 Blood filtering device

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1587401A (en) * 2004-09-15 2005-03-02 黄耀江 Process for preparing thrombase
CN101067132A (en) * 2006-11-17 2007-11-07 马忠仁 Thrombin extracting and separating process
CN101306353A (en) * 2008-02-02 2008-11-19 中国人民解放军南京军区南京总医院 Heparin affinity column and preparation method and use thereof
CN101979532A (en) * 2010-10-13 2011-02-23 江苏省江大绿康生物工程技术研究有限公司 Method for comprehensively using pig blood
CN203763551U (en) * 2013-12-26 2014-08-13 中原大学 Blood filtering device

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MANSETH E ET AL: "Developing a fish meat-binding agent: purification of salmon thrombin", 《JOURNAL OF FOOD SCIENCE》 *
朱蒙 等: "离子交换层析与亲和层析纯化猪凝血酶的比较", 《天然产物研究与开发》 *
王海亭 等: "凝血酶的亲和层析纯化技术", 《青岛化工学院学报》 *
肖丽霞 等: "猪凝血酶的分离纯化及部分性质研究", 《江苏农业研究》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105039273A (en) * 2015-07-13 2015-11-11 青岛康大食品有限公司 Method for purifying and preparing superoxide dismutase in rabbit blood
CN105039295A (en) * 2015-09-15 2015-11-11 上海洲跃生物科技有限公司 Method for preparing human thrombin from cold-removing glue plasma
CN108473530A (en) * 2015-10-15 2018-08-31 普莱泽玛科技有限公司 The method for extracting proteins from blood plasma
CN108473530B (en) * 2015-10-15 2022-12-23 普莱泽玛科技有限公司 Method for extracting protein from blood plasma
CN108660126A (en) * 2018-06-08 2018-10-16 博雅生物制药集团股份有限公司 A kind of preparation process of freeze dried human zymoplasm

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