CN104262469A - Method for separating and purifying pertactin - Google Patents
Method for separating and purifying pertactin Download PDFInfo
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- CN104262469A CN104262469A CN201410516831.4A CN201410516831A CN104262469A CN 104262469 A CN104262469 A CN 104262469A CN 201410516831 A CN201410516831 A CN 201410516831A CN 104262469 A CN104262469 A CN 104262469A
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Abstract
The invention discloses a method for separating and purifying pertactin. The method comprises the following steps: S1, sample treatment, namely, collecting a bordetella pertussis thallus, re-suspending, extracting and centrifuging the thallus to remove precipitate, carrying ultrafiltration and concentration on the supernate, and regulating the pH value being 7.0; S2, primary elution, namely, loading the supernate on a CaptoAdhere chromatographic column, eluting by a buffer solution, and collecting the eluent; S3, dialysis, namely, dialyzing and desalting the eluent in 15-25mM of a NaAc solution of which the pH is 4.5-5.5 to obtain dialysate; S4, secondary elution, namely, loading the dialysate on a CaptoSPImRes chromatographic column, eluting and collecting the eluent which is the purified pertactin. According to the method, the thallus of a bordetella pertussis nutrient solution is separated and purified by adopting the CaptoAdhere chromatographic column and the CaptoSPImRes chromatographic column, the prepared antigen protein pertactin has the advantages of exact component and controllable quality, and the purifying method has the advantages of high purification speed and low cost, and is simple to operate.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method of separation and purification PRN.
Background technology
Whooping cough (pertussis, whooping cough) be that the one caused by Gram-negative bordetella pertussis has strong communicable respiratory system disease, classical symptom is unexpected paroxysmal paroxysmal spasmodic cough, and with inspiratory coda or with vomiting, newborn infant and infant patient can cause breathlessness and cyanosis.Whooping cough at the initial stage of a disease infectivity is extremely strong, not easily diagnoses, and therefore carrying out vaccine inoculation is prevention and corntrol Whooping cough the most effective most economical means.
Its morbid substance of bordetella pertussis mainly comprises Toxins, pertussis (Pertussis Toxin, PT), filamentous hemagglutinin (Filamentous Hemagglutinin, FHA), PRN (Pertactin, PRN), agglutinogen (Agglutinogens, AGGs), adenylate cyclase toxin (Adenylate Cyclase Toxin, ACT), tracheal cell toxin (Tracheal Cytotoxin, and heat-labile toxin (Heat-labile toxin TCT), the various bioactivators such as HLT), these biologically active substances bordetella pertussis pathogenic effects and cause host to the immune response important role of disease.Pertussis vaccine in the market mainly contains whole cell pertussis vaccine and acellular pertussis vaccine two kinds, the latter belongs to the upgrading products of pertussis vaccine, only containing the Effective Antigens of purifying, baa has Pertussis somatic for it, therefore greatly reduce the untoward reaction after injection and remain good immune effect, being therefore widely used.
The development trend of acellular pertussis vaccine; remove useless by means of purification and cause the component (as HLT, TCT and ACT) of side reaction; retain and the antigenic component (as PT, FHA and PRN etc.) with protective immunity effect of purifying, traditional acellular pertussis vaccine antigen preparation technology can be divided into following two kinds:
1. centrifugal copurification technique: the centrifugal copurification technique that China and major part manufacturer of Japan adopt, namely after inoculum results, obtain refining antigen by ammonium sulfate precipitation, high level salt solution lixiviate and sucrose density gradient centrifugation, then utilize glutaraldehyde solution or formaldehyde solution to be toxoid by antigen detoxification; But this kind of method required equipment investment is huge, and the product purity of this explained hereafter is not high enough, productive rate is also lower, is not suitable for scale operation;
2. column chromatography: comprise following several: (1) hydroxyapatite, haptoglobin column chromatography and gel filtration method; (2) blue glue affinity chromatography and Pp63 glycophosphoproteins affinity chromatography; (3) blue glue affinity chromatography and hydroxylapatite chromatography; (4) perlite chromatography and hydroxyapatite column; Although use these techniques all can obtain purer pertussis antigen PT and FHA, but (1), (2), (3) technique have its inevitable problem, such as: blue glue affinity chromatography, Pp63 glycophosphoproteins affinity chromatography, haptoglobin affinity chromatography, the conjugated group of chromatographic stuffing is easily degraded and affects vaccine quality.In addition, in elutriant containing toxic substance or elution requirement too harsh, these all can directly affect antigen yield and quality; Technique (4) is though can obtain PT and FHA of higher degree and productive rate, but vaccine culture is after perlite column chromatographic isolation and purification, the purity of PT and FHA can only reach 62%-68%, also will through hydroxyapatite column separation and purification, the purity of PT and FHA just can reach about 90%, hydroxyapatite is expensive, and PT and FHA of this step about 10% loses.And acellular pertussis vaccine antigen preparation technology is about PT and FHA mostly, little to the report of PRN separation and purification.
Summary of the invention
The object of the invention is to the shortcoming overcoming prior art, a kind of method of separation and purification PRN is provided, the present invention adopts Capto Adhere chromatography column, Capto SP ImRes chromatography column carries out separation and purification to the thalline of bordetella pertussis nutrient solution, gained antigen protein PRN definite ingredients, quality controllable, purification process has simple to operate, that purifying speed is fast, cost is low advantage.
Object of the present invention is achieved through the following technical solutions: a kind of method of separation and purification PRN, and it comprises the following steps:
S1. sample preparation: collect bordetella pertussis thalline, thalline through resuspended, lixiviate, centrifugal segregation precipitation, by supernatant liquor with 30KD molecular weight cut-off film bag ultrafiltration and concentration to 1/15 ~ 1/10 of original volume, and adjust pH is to 7.0;
S2. a wash-out: be splined on by supernatant liquor on the Capto Adhere chromatography column that balances with phosphate buffered saline buffer, use A liquid, B liquid, C liquid, D liquid wash-out successively, collects the elutriant of D liquid, is PRN crude product;
Wherein, described A liquid is pH7.0,15 ~ 25mM PB, 0.7 ~ 1.3M NaCl, B liquid is pH4 ~ 5,15 ~ 25mM NaAC, 0.7 ~ 1.3M NaCl, C liquid are pH4 ~ 5,15 ~ 25mM NaAC, 0.1 ~ 0.5M NaCl, D liquid is pH4 ~ 5,15 ~ 25mM NaAC, 0.03 ~ 0.07M NaCl;
S3. dialyse: PRN crude product is carried out dialysis desalting in pH 4.5 ~ 5.5,15 ~ 25mM NaAc solution, obtains dialyzate;
S4. two wash-outs: dialyzate is splined on the Capto SP ImRes chromatography column balanced, carries out wash-out by pH 4.5 ~ 5.5,15 ~ 25mM NaAc, 0.1 ~ 0.2M NaCl solution, collect the PRN that elutriant is purifying.
Further, the concrete operations of sample preparation described in step S1 are: bordetella pertussis nutrient solution is cooled to 2 ~ 8 DEG C, centrifugal segregation supernatant liquor, collect thalline, by resuspended in pH8 ~ 9,90 ~ 110mM Tris, 130 ~ 170mM NaCl damping fluid for the thalline collected, every gram of thalline adds 8 ~ 12ml damping fluid, by supernatant liquor with 30KD molecular weight cut-off film bag ultrafiltration and concentration to 1/15 ~ 1/10 of original volume, the acetic acid adjust pH with 40 ~ 60% is to 7.0.
Further, the SP of Capto described in step S4 ImRes chromatography column adopts 20mM NaAC, the solution equilibria of pH5.0.
The present invention has the following advantages: the present invention adopts Capto Adhere chromatography column, Capto SP ImRes chromatography column carries out separation and purification to the thalline of bordetella pertussis nutrient solution, gained antigen protein PRN definite ingredients, quality controllable, the purity of separation and purification PRN reaches 99%, and purification process has simple to operate, that purifying speed is fast, cost is low advantage.
Accompanying drawing explanation
Fig. 1 is the chromatography collection of illustrative plates of the present invention's wash-out;
Fig. 2 is the chromatography collection of illustrative plates of secondary wash-out of the present invention;
Fig. 3 is the PRN purity check electrophorogram of separation and purification of the present invention.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described, and protection scope of the present invention is not limited to the following stated.
embodiment 1:a method for separation and purification PRN, it comprises the following steps:
S1. sample preparation: bordetella pertussis nutrient solution is cooled to 2 ~ 8 DEG C, centrifugal segregation supernatant liquor, collect thalline, by resuspended in pH8,90mM Tris, 130mM NaCl damping fluid for the thalline collected, every gram of thalline adds 8ml damping fluid, by supernatant liquor with 30KD molecular weight cut-off film bag ultrafiltration and concentration to 1/15 of original volume, the acetic acid adjust pH with 40% is to 7.0;
S2. a wash-out: supernatant liquor is splined on the Capto Adhere chromatography column that balances with phosphate buffered saline buffer, use A liquid (pH7.0,15mM PB, 0.7M NaCl), B liquid (pH4 successively, 15mM NaAC, 0.7M NaCl), C liquid (pH4,15mM NaAC, 0.1M NaCl), D liquid (pH4,15mM NaAC, 0.03M NaCl) wash-out, collect the elutriant of D liquid, be PRN crude product, the chromatography collection of illustrative plates of a wash-out as shown in Figure 1;
S3. dialyse: PRN crude product is carried out dialysis desalting in pH 4.5,15mM NaAc solution, obtains dialyzate;
S4. two wash-outs: dialyzate is splined on and uses 20mM NaAC, the Capto SP ImRes chromatography column that the solution equilibria of pH5.0 is good, wash-out is carried out by pH 4.5,15mM NaAc, 0.1M NaCl solution, collect the PRN that elutriant is purifying, the chromatography collection of illustrative plates of secondary wash-out as shown in Figure 1.
embodiment 2:a method for separation and purification PRN, it comprises the following steps:
S1. sample preparation: bordetella pertussis nutrient solution is cooled to 8 DEG C, centrifugal segregation supernatant liquor, collect thalline, by resuspended in pH9,110mM Tris, 170mM NaCl damping fluid for the thalline collected, every gram of thalline adds 12ml damping fluid, by supernatant liquor with 30KD molecular weight cut-off film bag ultrafiltration and concentration to 1/10 of original volume, the acetic acid adjust pH with 60% is to 7.0;
S2. a wash-out: supernatant liquor is splined on the Capto Adhere chromatography column that balances with phosphate buffered saline buffer, use A liquid (pH7.0,25mM PB, 1.3M NaCl), B liquid (pH5 successively, 25mM NaAC, 1.3M NaCl), C liquid (pH5,25mM NaAC, 0.5M NaCl), D liquid (pH5,25mM NaAC, 0.07M NaCl) wash-out, collect the elutriant of D liquid, be PRN crude product, the chromatography collection of illustrative plates of a wash-out as shown in Figure 1;
S3. dialyse: PRN crude product is carried out dialysis desalting in pH5.5,25mM NaAc solution, obtains dialyzate;
S4. two wash-outs: dialyzate is splined on and uses 20mM NaAC, the Capto SP ImRes chromatography column that the solution equilibria of pH5.0 is good, wash-out is carried out by pH 5.5,25mM NaAc, 0.2M NaCl solution, collect the PRN that elutriant is purifying, the chromatography collection of illustrative plates of secondary wash-out as shown in Figure 2.
embodiment 3:a method for separation and purification PRN, it comprises the following steps:
S1. sample preparation: bordetella pertussis nutrient solution is cooled to 4 DEG C, centrifugal segregation supernatant liquor, collect thalline, by resuspended in pH8.3,100mM Tris, 140mM NaCl damping fluid for the thalline collected, every gram of thalline adds 10ml damping fluid, by supernatant liquor with 30KD molecular weight cut-off film bag ultrafiltration and concentration to 1/12 of original volume, the acetic acid adjust pH with 48% is to 7.0;
S2. a wash-out: supernatant liquor is splined on the Capto Adhere chromatography column that balances with phosphate buffered saline buffer, use A liquid (pH7.0,18mM PB, 1.0M NaCl), B liquid (pH4.5 successively, 20mM NaAC, 1.0M NaCl), C liquid (pH4.5,20mM NaAC, 0.2M NaCl), D liquid (pH4.5,20mM NaAC, 0.04M NaCl) wash-out, collect the elutriant of D liquid, be PRN crude product, the chromatography collection of illustrative plates of a wash-out as shown in Figure 1;
S3. dialyse: PRN crude product is carried out dialysis desalting in pH 5.0,18mM NaAc solution, obtains dialyzate;
S4. two wash-outs: dialyzate is splined on and uses 20mM NaAC, the Capto SP ImRes chromatography column that the solution equilibria of pH5.0 is good, wash-out is carried out by pH 5.0,18mM NaAc, 0.15M NaCl solution, collect the PRN that elutriant is purifying, the chromatography collection of illustrative plates of secondary wash-out as shown in Figure 2.
embodiment 4:a method for separation and purification PRN, it comprises the following steps:
S1. sample preparation: bordetella pertussis nutrient solution is cooled to 6 DEG C, centrifugal segregation supernatant liquor, collect thalline, by resuspended in pH8.7,108mM Tris, 160mM NaCl damping fluid for the thalline collected, every gram of thalline adds 11ml damping fluid, by supernatant liquor with 30KD molecular weight cut-off film bag ultrafiltration and concentration to 1/14 of original volume, the acetic acid adjust pH with 53% is to 7.0;
S2. a wash-out: supernatant liquor is splined on the Capto Adhere chromatography column that balances with phosphate buffered saline buffer, use A liquid (pH7.0,22mM PB, 1.2M NaCl), B liquid (pH4.8 successively, 18mM NaAC, 1.2M NaCl), C liquid (pH4.8,23mM NaAC, 0.3M NaCl), D liquid (pH4.3,23mM NaAC, 0.06M NaCl) wash-out, collect the elutriant of D liquid, be PRN crude product, the chromatography collection of illustrative plates of a wash-out as shown in Figure 1;
S3. dialyse: PRN crude product is carried out dialysis desalting in pH 5.2,18mM NaAc solution, obtains dialyzate;
S4. two wash-outs: dialyzate is splined on and uses 20mM NaAC, the Capto SP ImRes chromatography column that the solution equilibria of pH5.0 is good, wash-out is carried out by pH 4.8,23mM NaAc, 0.18M NaCl solution, collect the PRN that elutriant is purifying, the chromatography collection of illustrative plates of secondary wash-out as shown in Figure 2.
embodiment 5:purity check (SDS-PAGE)
1. experimental technique: SDS-PAGE argentation.
1.1 sample preparation: measuring samples is that the PRN (PRN) of embodiment 3 from purifying is first by diluted sample, generally measuring samples can be diluted to every milliliter of 0.3-0.5 milligram, loading sample contains the product to be checked of 3/4ths, and the sample treatment liquid of 1/4th, does not need water-bath.Applied sample amount is generally per pass containing measuring samples PRN 2 microgram, Whooping cough standard substance (PRN) albumen 2-3 microgram, molecular weight standards albumen (Maker) 2 microgram.
1.2 electrophoresis also dye: this experiment adopts vertical electrophoresis, starts concentrated glue voltage lower (70-80V, about 20 minutes), voltage high (150-200V) during separation gel, the about 60-80 minute of whole process electrophoresis time.When tetrabromophenol sulfonphthalein forward position arrives (anode) bottom electrophoresis chamber, cut off the electricity supply, open electrophoresis chamber upper cover, take out electrophoresis chamber support and the liquid outwelled in inside groove, carefully take off glass plate and two panels glass plate is separated, lightly the concentrated glue on glass plate separated with separation gel and cast out concentrated glue part, separation gel being taken off carefully put into and off-the-shelfly to have in the container of stationary liquid (or large plate) and start to dye, dyeing as argentation.
2. experimental result: reduction running gel protein through scanning, calculate measuring samples PRN relative to band account for the per-cent of total protein.
Electrophorogram is as shown in Figure 3:
In figure: M:maker;
PRN1 is the PRN crude product of a wash-out;
PRN2 is the PRN of the purifying of secondary wash-out.
as shown in Figure 3: PRN molecular weight is 66KD, electrophoretogram display is consistent with bibliographical information, and the PRN purity obtained through the inventive method reaches 99%.
Claims (3)
1. a method for separation and purification PRN, is characterized in that, it comprises the following steps:
S1. sample preparation: collect bordetella pertussis thalline, thalline through resuspended, lixiviate, centrifugal segregation precipitation, by supernatant liquor with 30KD molecular weight cut-off film bag ultrafiltration and concentration to 1/15 ~ 1/10 of original volume, and adjust pH is to 7.0;
S2. a wash-out: be splined on by supernatant liquor on the Capto Adhere chromatography column that balances with phosphate buffered saline buffer, use A liquid, B liquid, C liquid, D liquid wash-out successively, collects the elutriant of D liquid, is PRN crude product;
Wherein, described A liquid is pH7.0,15 ~ 25mM PB, 0.7 ~ 1.3M NaCl, B liquid is pH4 ~ 5,15 ~ 25mM NaAC, 0.7 ~ 1.3M NaCl, C liquid are pH4 ~ 5,15 ~ 25mM NaAC, 0.1 ~ 0.5M NaCl, D liquid is pH4 ~ 5,15 ~ 25mM NaAC, 0.03 ~ 0.07M NaCl;
S3. dialyse: PRN crude product is carried out dialysis desalting in pH 4.5 ~ 5.5,15 ~ 25mM NaAc solution, obtains dialyzate;
S4. two wash-outs: dialyzate is splined on the Capto SP ImRes chromatography column balanced, carries out wash-out by pH 4.5 ~ 5.5,15 ~ 25mM NaAc, 0.1 ~ 0.2M NaCl solution, collect the PRN that elutriant is purifying.
2. the method for a kind of separation and purification PRN as claimed in claim 1, it is characterized in that, the concrete operations of sample preparation described in step S1 are: bordetella pertussis nutrient solution is cooled to 2 ~ 8 DEG C, centrifugal segregation supernatant liquor, collect thalline, by resuspended in pH8 ~ 9,90 ~ 110mM Tris, 130 ~ 170mM NaCl damping fluid for the thalline collected, every gram of thalline adds 8 ~ 12ml damping fluid, by supernatant liquor with 30KD molecular weight cut-off film bag ultrafiltration and concentration to 1/15 ~ 1/10 of original volume, the acetic acid adjust pH with 40 ~ 60% is to 7.0.
3. the method for a kind of separation and purification PRN as claimed in claim 1, is characterized in that, the SP of Capto described in step S4 ImRes chromatography column adopts 20mM NaAC, the solution equilibria of pH5.0.
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| CN201410516831.4A CN104262469A (en) | 2014-09-30 | 2014-09-30 | Method for separating and purifying pertactin |
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| CN201410516831.4A CN104262469A (en) | 2014-09-30 | 2014-09-30 | Method for separating and purifying pertactin |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584958A (en) * | 2012-02-08 | 2012-07-18 | 北京天坛生物制品股份有限公司 | Purification method for 69KD outer membrane protein of pertussis bacillus |
| EP2592137A1 (en) * | 2011-11-11 | 2013-05-15 | Novartis AG | Fermentation media free of animal-derived components for production of diphtheria toxoids suitable for human vaccine use |
| CN103242434A (en) * | 2012-02-08 | 2013-08-14 | 长春百克生物科技股份公司 | Method for preparing acellular pertussis vaccine |
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- 2014-09-30 CN CN201410516831.4A patent/CN104262469A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2592137A1 (en) * | 2011-11-11 | 2013-05-15 | Novartis AG | Fermentation media free of animal-derived components for production of diphtheria toxoids suitable for human vaccine use |
| CN102584958A (en) * | 2012-02-08 | 2012-07-18 | 北京天坛生物制品股份有限公司 | Purification method for 69KD outer membrane protein of pertussis bacillus |
| CN103242434A (en) * | 2012-02-08 | 2013-08-14 | 长春百克生物科技股份公司 | Method for preparing acellular pertussis vaccine |
Non-Patent Citations (1)
| Title |
|---|
| 田阳等: ""百日咳毒素、丝状血凝素和黏附素的纯化"", 《中国生物制品学杂志》 * |
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Application publication date: 20150107 |