CN1156726A - Process for purifying bactericidal power/permeability increasing protein from big blood - Google Patents
Process for purifying bactericidal power/permeability increasing protein from big blood Download PDFInfo
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- CN1156726A CN1156726A CN 96117850 CN96117850A CN1156726A CN 1156726 A CN1156726 A CN 1156726A CN 96117850 CN96117850 CN 96117850 CN 96117850 A CN96117850 A CN 96117850A CN 1156726 A CN1156726 A CN 1156726A
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Abstract
A process for extracting and purifying bactericidal permeable incremental-protein (BPI) from pig blood includes such steps as freezing PMNL purified from pig blood to obtain coarse extract liquid of BPI, culturing by colibacillus as binding bacteria, centrifugal separation to obtain binding thallus, separating protein from thallus, dialysis and centrifugal separation to obtain purified BPI. Its advantages include simple apparatus, easily controlled condition, less pollution and low cost.
Description
The present invention relates to a kind of proteinic extracting and purifying method, especially a kind of purifying bactericidal properties/power/permeability increasing protein method of (being called for short BPI) of from pig blood, extracting.
Known BPI is the positive protein matter in the Mammals neutrophil leucocytes such as a kind of people of being present in and rabbit (being called for short PMNL), it is a kind of strong bacteriocidal substance, have and specially kill Gram-negative bacteria and neutralize endotoxic activity, therefore, people's desire is extracted BPI from the PMNL of people and rabbit, clinical in the hope of being applied to, however traditional BPI method of purification is: 1. and the purifying PMNL with people or rabbit adopts polishing to destroy cytolemma, and gets crude extract with the extracting of 0.16N sulfuric acid; 2. be the BPI that dextran G-75 or G-50, dextran C-50 and dextran G-100 obtain purifying with crude extract through three column chromatographies.This extraction method of purification has weak point: 1. raw material resources scarcity is not easy to obtain; 2. adopt polishing to destroy cytolemma, dustiness height; 3. through three column chromatographies, workload is big, and appointed condition requires high and wayward, consuming timely reaches 1-2 week.For above-mentioned reasons, make its cost height, be not easy to obtain, hindered its value performance.
The object of the present invention is to provide the extraction method of purification of a kind of simple, quick, economic BPI.
Find after deliberation, have BPI among the PMNL of pig blood, it is a positively charged protein, and the electronegative part of intracellular toxin of it and G-bacterium adventitia has very high avidity, this avidity substantially exceeds the avidity with other materials, and the BPI in this combination can be by high density Mg
2+Replace to get off.
In view of above-mentioned viewpoint, the present invention is achieved in that the method for purifying bactericidal/power/permeability increasing protein in the boar blood is at first producing the BPI crude extract with the PMNL employing cold method of purifying in the pig blood; Again with intestinal bacteria as hatch in conjunction with bacterium, centrifugal obtaining in conjunction with thalline; Then from conjunction with isolating protein the thalline; Dialyse the centrifugal purifying BPI that gets at last.
It is freezing that the producing of wherein said BPI crude extract is meant that the BPI with purifying carries out, and multigelation is more than three times or three times, and to get PH with the extracting of 0.16N sulfuric acid be 4.0 BPI crude extract.
Wherein speed is meant the BPI crude extract that intestinal bacteria 15 bacterium is placed PH4.0 in conjunction with the method for making of thalline, and making its J5 bacterium final concentration is 5 * 10
8Cfu/ml is hatched 10-15min under 37 ℃ of conditions, centrifugal collection is in conjunction with thalline.
The condition of wherein said isolated protein is for placing the damping fluid of PH4.0 with gained in conjunction with thalline, and making in conjunction with the thalline final concentration is 5 * 10
9Cfu/m1,15min is hatched in 37 ℃ of vibrations, and at 4 ℃, centrifugal 20min gets supernatant under 23000 * g condition, and this supernatant is isolated protein.
Wherein said dialysis, centrifugal condition are that 2000 times were dialysed 24 hours in the dialyzate of above-mentioned supernatant volume, and at 4 ℃, centrifugal 20min gets the BPI of purifying under 23000 * g condition.
Wherein said PH4.0 damping fluid in conjunction with indication in the thalline method for making is 200mMMgCl
2/ 10mMHAc/NaAc.
Wherein said dialyzate is meant the 10mMHAC/NaPH4.0 damping fluid.
The equipment that this method adopts is simple, and condition is easy to control, weak point consuming time, and the time can be above 48 hours; Adopt cold method to destroy cytolemma, dustiness is reduced to minimum, and saved manpower again than polishing; Pig blood is cheap and easy to get, make it all reduce cost from starting material and equipment, in sum, this is the method for a kind of simple, quick, economic purification BPI, reduced the cost of BPI on the whole, and the BPI that obtains of this method after tested its purifying multiple and purity can satisfy clinical demand, for its clinical application is laid a good foundation.
The invention will be further described below in conjunction with embodiment:
The method of purifying bactericidal/power/permeability increasing protein is in the one boar blood: at first the PMNL with purifying in the pig blood adopts cold method to produce the BPI crude extract; Again with intestinal bacteria as hatch in conjunction with bacterium, centrifugal obtaining in conjunction with thalline; Then from conjunction with isolating protein the thalline; Dialyse the centrifugal purifying BPI that gets at last.
It is freezing that the producing of wherein said BPI crude extract is meant that the PMNL with purifying from pig blood carries out, and freezing temp is 0 ℃ of multigelation three times, and to get PH with the extracting of 0.16N sulfuric acid be 4.0 BPI crude extract.
Wherein said method for making in conjunction with thalline is meant the BPI crude extract that intestinal bacteria J5 bacterium is placed PH4.0, makes J
5The bacterium final concentration is 5 * 10
8Cfu/ml is hatched 10-15min under 37 ℃ of conditions, with whizzer under 1000rpm * 10min condition centrifugal collection in conjunction with thalline.
The condition of wherein said isolated protein is for placing gained the 200mMMg (Gl of PH4.0 in conjunction with thalline
2In/10mMHAc/NaHAc the damping fluid, making in conjunction with the thalline final concentration is 5 * 10
9Cfu/ml, 15min is hatched in 37 ℃ of vibrations, and at 4 ℃, centrifugal 20min gets supernatant under 23000 * g condition, and this supernatant is isolated protein.
Wherein said dialysis, centrifugal condition are that 2000 times were dialysed 24 hours in the volume 10mMHAc/NaACPH4.0 of above-mentioned supernatant damping fluid, and at 4 ℃, centrifugal 20min gets the BPI of purifying under 23000 * g condition.
The BPI of gained is compared with the BPI test that PMNL from pig blood purifies by traditional column chromatography, must table 1:
Two kinds of method contrast tables of table 1.
| The albumen amount of obtaining (%) | Fungicidal activity * | In and the intracellular toxin ability | The purifying multiple | |
| The present invention | ??????0.1 | ????0.75 | ??????30.9 | ???103 |
| The tradition method | ??????0.1 | ????0.08 | ??????51.0 | ???170 |
*: fungicidal activity is for killing 90%3 * 10
7The milligram number of intestinal bacteria desirable proteins.
Claims (7)
1. the method for purifying bactericidal/power/permeability increasing protein in the boar blood is characterized in that: at first the PMNL employing cold method of purifying in the pig blood is produced the BPI crude extract; Again with intestinal bacteria as hatch in conjunction with bacterium, centrifugal obtaining in conjunction with thalline; Then from conjunction with isolating protein the thalline; Dialyse the centrifugal purifying BPI that gets at last.
2. the method for purifying bactericidal/power/permeability increasing protein in the pig blood as claimed in claim 1, it is characterized in that: the PMNL that is meant purifying in the pig blood that produces of described BPI crude extract carries out freezing, multigelation is more than three times or three times, and to get PH with the extracting of 0.16N sulfuric acid be 4.0 BPI crude extract.
3. the method for purifying bactericidal/power/permeability increasing protein in the pig blood as claimed in claim 1 is characterized in that: described method for making in conjunction with thalline is meant the BPI crude extract that intestinal bacteria 15 bacterium is placed PH4.0, and making J5 bacterium final concentration is 5 * 10
8Cfu/ml is hatched 10-15min under 37 ℃ of conditions, centrifugal collection is in conjunction with thalline.
4. the method for purifying bactericidal/power/permeability increasing protein in the pig blood as claimed in claim 1 is characterized in that: the condition of described isolated protein is for placing the damping fluid of PH4.0 with gained in conjunction with thalline, and making in conjunction with the thalline final concentration is 5 * 10
9Cfu/ml, 15min is hatched in 37 ℃ of vibrations, and at 4 ℃, centrifugal 20min gets supernatant under 23000 * g condition, and this supernatant is isolated protein.
5. the method for purifying bactericidal/power/permeability increasing protein in the pig blood as claimed in claim 1, it is characterized in that: described dialysis, centrifugal condition are for dialysing 24 hours in the dialyzate of above-mentioned supernatant volume 2000 times, at 4 ℃, centrifugal 20min gets the BPI of purifying under 23000 * g condition.
6. the method for purifying bactericidal/power/permeability increasing protein in the pig blood as claimed in claim 4 is characterized in that: described PH4.0 damping fluid is 200mMMgCl
2/ 10mMHAc/NaAc.
7. the method for purifying bactericidal/power/permeability increasing protein in the pig blood as claimed in claim 5 is characterized in that: described dialyzate is the 10mMHAc/NaAcPH4.0 damping fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN96117850A CN1062564C (en) | 1996-12-20 | 1996-12-20 | Process for purifying bactericidal power/permeability increasing protein from big blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN96117850A CN1062564C (en) | 1996-12-20 | 1996-12-20 | Process for purifying bactericidal power/permeability increasing protein from big blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1156726A true CN1156726A (en) | 1997-08-13 |
| CN1062564C CN1062564C (en) | 2001-02-28 |
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ID=5124666
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96117850A Expired - Fee Related CN1062564C (en) | 1996-12-20 | 1996-12-20 | Process for purifying bactericidal power/permeability increasing protein from big blood |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586162A (en) * | 2011-11-29 | 2012-07-18 | 怀化学院 | Genetic engineering bacterium for producing recombined pig sterilization protein and construction and application thereof |
| CN106164344A (en) * | 2014-02-07 | 2016-11-23 | 韩国生命工学研究院 | The method using protein fusion display technique high throughput assay Substance Interactions |
| CN108660125A (en) * | 2018-05-14 | 2018-10-16 | 武汉博百欧生物科技有限公司 | A method of extracting neutrophil leucocyte azurophilic granule in transfusing blood waste from human body |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5420019A (en) * | 1993-02-02 | 1995-05-30 | Xoma Corporation | Stable bactericidal/permeability-increasing protein muteins |
| CN100396695C (en) * | 1993-03-12 | 2008-06-25 | 爱克索马技术有限公司 | Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof |
-
1996
- 1996-12-20 CN CN96117850A patent/CN1062564C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586162A (en) * | 2011-11-29 | 2012-07-18 | 怀化学院 | Genetic engineering bacterium for producing recombined pig sterilization protein and construction and application thereof |
| CN106164344A (en) * | 2014-02-07 | 2016-11-23 | 韩国生命工学研究院 | The method using protein fusion display technique high throughput assay Substance Interactions |
| CN108660125A (en) * | 2018-05-14 | 2018-10-16 | 武汉博百欧生物科技有限公司 | A method of extracting neutrophil leucocyte azurophilic granule in transfusing blood waste from human body |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1062564C (en) | 2001-02-28 |
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Owner name: THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY M Free format text: FORMER OWNER: ZHOU HONG Effective date: 20040702 |
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Effective date of registration: 20040702 Address after: Chongqing city Shapingba street 400038 gaotanyan No. 30 Patentee after: No.1 Attached Hospital, No.3 Military Medical College PLA. Address before: 630038 Burn Research Institute, Southwest Hospital, Chongqing, Sichuan, China Patentee before: Zhou Hong |
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