CN105175517A - Separation and purification method for pertussis antigen - Google Patents
Separation and purification method for pertussis antigen Download PDFInfo
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- CN105175517A CN105175517A CN201510595876.XA CN201510595876A CN105175517A CN 105175517 A CN105175517 A CN 105175517A CN 201510595876 A CN201510595876 A CN 201510595876A CN 105175517 A CN105175517 A CN 105175517A
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Abstract
The invention provides a separation and purification method for a pertussis antigen. The separation and purification method comprises the steps of with diatomite and hydroxylapatite as column chromatography materials, after concentrating, regulating and conducting a bordetella pertussis fermentation solution, enriching the pertussis antigen by using the diatomite, and then, purifying by using the hydroxylapatite to obtain pertussis toxin (PT) and filamentous hemagglutinin (FHA). Proven through SDS-PAGE electrophoresis detection, the purities of both a PT protein and an FHA protein are higher than 95%. The method provided by the invention is simple in operation and capable of obtaining a high-purity antigen, not only greatly reducing the production cost, but also remarkably improving the quality of a product and increasing the yield of the product so as to be the simple and rapid separation and purification method for the pertussis antigen; in addition, the economic benefit is high, and the market application prospect is wide.
Description
Technical field
The present invention relates to technical field of biological product preparation, specifically, relate to a kind of separation purification method of pertussis antigen.
Background technology
Whooping cough below four years old in children population M & M all very high, early stage various countries use whole cell pertussis vaccine to prevent, but whole cell pertussis vaccine may cause the side reaction of the symptom such as fever, vomiting, injection site redness in various degree, individual other also may produce more serious cranial nerve disease, even dead.Therefore some country, as: Britain, Sweden and Japan etc. once stopped inoculation whole cell pertussis vaccine.But it is popular to there occurs Whooping cough subsequently.Therefore last century the eighties, first Japan prepared acellular pertussis vaccine and substituted whole-cell vaccines, it is the Effective Antigens albumen being extracted bordetella pertussis, after filamentous hemagglutinin (FHA) and Toxins, pertussis (PT), the non-whole-cell vaccines prepared after detoxification treatment again, the side reaction of vaccine greatly reduces.
Pertussis vaccine, from implementation plan immunity in 1985, is included in the vaccine of first planned immunization by China, and pertussal sickness rate is sharply declined.The production of current domestic widely used domestic DTaP vaccine is the ammonium sulfate copurification technology adopted; the Whooping cough protective antigen of preparation belongs to hybrid antigen; content, the ratio of its effective constituent are unstable; differ greatly between batch; simultaneously because purification means fall behind; cause impurity component too much, side reaction is still comparatively serious.The backwardness of the purifying process level of pertussis antigen has seriously limited the use of domestic tin-free self-polishing anti-fou ling paint, also directly affects the enforcement of National immunization Program.Therefore new purifying process is adopted to prepare the technology of acellular whooping cough component vaccine extremely important.
In past 20 years, North America and European Countries adopt column chromatography purification technology new generation acellular pertussis vaccine one after another, preparation technology comprises following several: hydroxyapatite, haptoglobin column chromatography and gel filtration method, blue glue affinity chromatography and Pp63 glycophosphoproteins affinity chromatography, blue glue affinity chromatography and hydroxylapatite chromatography.But these methods of the prior art all exist respective weak point, 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 toxicant or elution requirement too harsh, these all can directly affect antigen yield and quality.Moreover, because the medium for purifying is very expensive, add production cost, be unfavorable for production and the extra earning of vaccine enterprise.Therefore, provide a kind of method of separation and purification pertussis antigen simple to operate, with low cost extremely important and urgent.
Summary of the invention
The object of this invention is to provide a kind of pertussis antigen separation purification method.
For realizing object of the present invention, the separation purification method of a kind of pertussis antigen provided by the invention, comprises the following steps:
(1) diatomite is loaded in chromatography column;
(2) bordetella pertussis fermented liquid centrifugal after get supernatant liquor, be the 5%-10% of original volume by supernatant liquor ultrafiltration and concentration, and adjust conductance;
(3) concentrated supernatant liquor loading, priority PB buffered soln, containing the PB buffered soln of TritonX-100, PB buffered soln, chromatography column to be balanced and wash-out foreign protein, again with the PB buffered soln elution chromatography post of 0.15-0.25M containing NaCl, obtain elutriant 1;
(4) with the PB buffered soln containing 0.5-1.0MNaCl, wash-out is carried out to chromatography column again and obtain elutriant 2;
(5) elutriant 1 is through hydroxyapatite chromatography post, and collection penetrates liquid and is Toxins, pertussis;
(6) elutriant 2 is after the absorption of hydroxyapatite chromatography post, first uses the non-associated proteins of PB eluant solution, then rinses chromatography column with the PB solution containing NaCl, and the elutriant of collection is Whooping cough filamentous hemagglutinin.
The diatomite particle diameter that the inventive method is selected is 40-300 μm.During use, available water for injection washing, removes floating matter and macrobead throw out.
Preferably, the diatomite particle diameter of the inventive method is 60-200 μm.
In the inventive method, after step (1) diatomite dress post, rinse chromatography column with the PB buffered soln of pH7.2-8.0,10-50mmol/L.
Wherein, step (2) fermented liquid centrifugal condition is 3500-7000r/min, centrifugal 30-60min.
Supernatant liquor after centrifugal adopts 10KDa hollow fiber column to carry out ultrafiltration.
Step of the present invention (2) adjustment electric conductivity value is 2-8ms/cm.Preferred electric conductivity value is 4-6ms/cm.
In one embodiment of the invention, be the conductance of the PB buffered soln adjustment ultrafiltration and concentration liquid adopting pH7.2-8.0,10-50mmol/L, PB buffered soln at the uniform velocity passes through diatomite chromatography column, flow velocity 50ml/min.
In the step (3) of the inventive method, supernatant liquor loading volume is 5-10 times of column volume, successively adopting 10-50mmol/LPB buffered soln to rinse chromatography column to OD value is 0-0.05, PB buffered soln containing 0.05%TritonX-100,10-50mmol/L rinses 5-10 column volume, it is 0-0.05 that the PB buffered soln of 10-50mmol/L rinses chromatography column to OD value, then adopts the PB buffered soln wash-out of NaCl, 10-50mmol/L containing 0.15-0.25M to obtain elutriant 1; Aforementioned PB buffered soln pH is pH7.2-8.0.
The step (4) of the inventive method rinses chromatography column with the 10-50mmol/LPB buffered soln containing 0.5-1.0MNaCl, pH7.2-8.0, and wash-out obtains elutriant 2.
In aforesaid method, step (3) and step (4) adopt the PB buffered soln containing different concns NaCl respectively, guarantee Toxins, pertussis (PT) and the thread blood clotting toxin (FHA) of Whooping cough to be eluted respectively.
In the inventive method, step (6) adopts pH7.2-8.0,10-50mmol/LPB to rinse chromatography column 5-10 column volume, adopt the 0.2-0.5MPB buffered soln elution chromatography post of 0.5-1.0MNaCl, pH7.2-8.0 to be 0-0.05 to OD value again, the elutriant of collection is Whooping cough filamentous hemagglutinin.
Present invention also offers the application of aforesaid method in vaccine preparation.
Further, described vaccine is pertussis vaccine or the vaccine containing pertussis antigen composition.
The inventive method adopts diatomite, hydroxyapatite as column chromatography material, after the process of bordetella pertussis fermentation liquor concentrated adjustment conductance, through diatomite enrichment pertussis antigen, then after hydroxyapatite purifying, obtain Toxins, pertussis (PT) and filamentous hemagglutinin (FHA).Through HPLC inspection, PT albumen and FHA purity of protein are all higher than 95%.The inventive method is simple to operate, the purity of antigen is high, pollution-free, dress column material diatomite cost is low, be easy to obtain, not only significantly reduce quality and the yield producing vaccine cost but also significantly improve pertussis antigen product, be a kind of simply, the separation purification method of pertussis antigen fast, be applicable to the large-scale need of production of batch production.
Accompanying drawing explanation
Fig. 1 be Ultraviolet Detector to diatomite Column eluate detection curve figure, diatomite purifying PT and FHA, in figure, peak 1 is for penetrating liquid foreign protein, and peak 2 is elutriant 1, and peak 3 is elutriant 2.
Fig. 2 is that Ultraviolet Detector penetrates the detection curve figure of liquid to elutriant 1 after hydroxyapatite chromatography post, and 1 is PT peak, and display purifying obtains PT.
Fig. 3 is the elutriant detection curve figure that Ultraviolet Detector is collected after hydroxyapatite chromatography post elutriant 2, and 1 is FHA peak, and display purifying obtains FHA.
Fig. 4 to be PT and the PT standard substance concentration of purifying be 12% separation gel carry out SDS-PAGE electrophorogram, swimming lane 1 is the PT of the inventive method purifying; Swimming lane 2 is molecular weight of albumen Marker, and molecular weight marker is respectively 14kd, 25kd, 30kd, 40kd, 62kd, 94kd, 150kd, 230kd from the bottom up; Swimming lane 3 is PT standard substance.
Fig. 5 to be PT and the PT standard substance concentration of purifying be 8% separation gel carry out SDS-PAGE electrophoresis, swimming lane 1 is the FHA of the inventive method purifying; Swimming lane 2 is molecular weight of albumen Marker, and molecular weight marker is respectively 40kd, 55kd, 70kd, 105kd, 220kd from the bottom up; Swimming lane 3 is FHA standard substance.
Fig. 6 is the Chinese hamster ovary celI bunch collection test of PT standard substance.
Fig. 7 is the Chinese hamster ovary celI bunch collection test of purifying PT.
Fig. 8 is Chinese hamster ovary celI bunch collection negative control experiments.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
Embodiment 1 pertussis antigen separation purification method
Select domestic diatomite (Filters limited liability company of radically reforming is created in Beijing), sieve and remove less and larger particles, obtain granulometric range 60 ~ 200 μm, water for injection is adopted to wash diatomite, remove floating matter and suspended substance, load in chromatography column (middle life magnificent (Beijing) Science and Technology Ltd., column diameter 100mm), loading height is 200mm, adopts the 10mmol/LPB (pH8.0) of 5 times to rinse chromatography column.
Open the seed bottle of Whooping cough Bordetella CS bacterial strain freeze-drying, purchased from National Institute for Food and Drugs Control, bacterial classification is transferred on improvement bag Jiang Shi substratum, cultivate 72 hours for 35.5 DEG C, be collected in and be transferred on gac half synthetic medium 35.5 DEG C and cultivate 48 hours, regather to be transferred on gac half synthetic medium and cultivate 48 hours, collect bacterial classification.The bacterial classification of collection is transferred in the seeding tank containing 30LS-S substratum, cultivates after 28 hours, transfer in the fermentor tank containing 240LS-S substratum, stir aerated culture 40 hours.After cultivation terminates, collected by centrifugation supernatant liquor, obtains the supernatant liquor of 240L.Centrifugal condition is 5000rpm, centrifugal 40min.
Supernatant liquor is concentrated into 12L through 10KDa hollow fiber column ultrafilter, adopts 10mmol/LPB (pH8.0) to change liquid to conductance 4 ~ 6ms/cm, through constant flow pump at the uniform velocity loading by diatomite chromatography column, flow velocity 50ml/min.Non-associated proteins is penetrated, and sees peak 2 in Fig. 1.
First using 10mmol/LPB (pH8.0) to balance chromatography column, is (0-0.05) to OD value; After 10mmol/LPB (pH8.0) the wash-out foreign protein containing 0.05%TritonX-100 again with 5 column volumes, then using 10mmol/LPB (pH8.0) to balance chromatography column, is (0-0.05) to OD value; Then obtain elutriant 1 with 10mmol/LPB (pH8.0) wash-out containing 0.2MNaCl, finally obtain elutriant 2 with 50mmol/LPB (pH8.0) wash-out containing 0.5MNaCl.Carry out on-line checkingi with the Ultraviolet Detector of 280nm wavelength to diatomite chromatography column wash-out, the detection curve of wash-out as shown in Figure 1.
Elutriant 1 is through hydroxyapatite chromatography post (Shanghai Jin Hua tomography devices factory), and will penetrate liquid and collect, the liquid that penetrates of collection is Toxins, pertussis (PT), sees Fig. 2.
Elutriant 2 is after the absorption of hydroxyapatite chromatography post, the 50mmol/LPB of 10 column volumes (pH8.0) is first used to carry out wash-out to unconjugated foreign protein, again with 0.4MPB (pH8.0) the elution chromatography post containing 1.0MNaCl, be 0-0.05 to OD value; The elutriant collected is Whooping cough filamentous hemagglutinin (FHA), sees Fig. 3.
Embodiment 2 diatomite specific adsorption PT and FHA
After fermented liquid after concentrated in 10ml embodiment 1 is regulated conductance to 4-6ms/cm with water for injection, fully mix with diatomite, 4 DEG C leave standstill 10 minutes.Remove supernatant liquor, diatomite precipitation 10ml10mmol/LPB (pH8.0) cleans three times, each after shaking up, leave standstill 10 minutes, remove supernatant, after having cleaned, in diatomite precipitation, add 10ml carry out wash-out containing the 50mmol/LPB (pH8.0) of 0.5MNaCl, type of elution leaves standstill 10 minutes for diatomite damping fluid being shaken up latter 4 DEG C, collects supernatant.Supernatant is through electrophoresis detection, and main component is PT and FHA.Result illustrates that diatomite is have specific adsorption effect to FHA, PT, and the wash-out of FHA, PT is relevant with salt concn.
Embodiment 3 purifying PT and FHA purity detecting
PT and FHA that purifying obtains is carried out SDS-PAGE electrophoresis, then measures purity of protein by the method for gel image scanning.
Test method is that the separation gel being first 12% by PT and the PT standard substance concentration of purifying carries out SDS-PAGE electrophoresis (electrophoresis result is shown in Fig. 4), FHA and the FHA standard substance concentration of purifying be 8% separation gel carry out SDS-PAGE electrophoresis (electrophoresis result is shown in Fig. 5), after electrophoresis, gel is carried out scanning analysis, calculates the purity of albumen.Result shows PT and the PT standard substance of purifying, FHA and the FHA standard substance of purifying have the same electrophoretogram, PT and the PT standard substance obtained by embodiment 1 method are described, FHA and FHA standard substance have consistence, purity check shows that the purity of PT and FHA that the method obtains all reaches more than 95%, far above Chinese Pharmacopoeia, acellular pertussis vaccine purity of protein is not less than to the requirement of 85%, the albumen of purifying gained can meet the requirement preparing pertussis vaccine.
Fig. 4 is the electrophorogram of PT and PT standard substance, and wherein swimming lane 1 is the PT obtained in embodiment 1, and swimming lane 2 is molecular weight of albumen Marker, swimming lane 3PT standard substance.Fig. 5 is the electrophorogram of FHA and FHA standard substance, and wherein swimming lane 1 is the FHA obtained in embodiment 1, and swimming lane 2 is molecular weight of albumen Marker, and swimming lane 3 is the FHA standard substance bought.
The hemagglutinative vigor of embodiment 4 purifying FHA measures
Take fresh goose blood 3ml, add 3ml0.85% physiological sodium chloride solution, centrifugal, abandon supernatant; Add 3ml0.85% physiological sodium chloride solution again, repeat 3 times.Add 9ml0.85% physiological sodium chloride solution again, be prepared into 10% goose blood suspension.
Get 0.85% physiological sodium chloride solution 50 μ l on 96 orifice plates, the supernatant concentrated solution of the bordetella pertussis fermented liquid that Example 1 obtains (20 × concentrated), penetrate liquid (PT) and each 50 μ l of elutriant (FHA) do doubling dilution respectively in 96 orifice plates, every hole adds 50 μ l goose blood, concussion mixing, static 20min experimental result is as table 1.Protein concentration in table 1 is detected by Lowry method, and result shows that the hemagglutination activity of the FHA of purifying is obviously better than supernatant concentrated solution, and the PT hemagglutination activity after purifying is very low.
Table 1 elution peak protein concentration and hemagglutination activity
The Chinese hamster ovary celI bunch collection test of embodiment 5 purifying PT
By the activity of the PT obtained in Chinese hamster ovary celI bunch collection testing inspection embodiment 1.PT standard substance, a Chinese hamster ovary celI bunch collection test method is provided by National Institute for Food and Drugs Control.
After cultured Chinese hamster ovary celI trysinization, add cell growth medium and carry out counting and being diluted to 8 × 10
4individual/ml, gets 100ul cell suspension and joins in 96 orifice plates, in cell, then respectively add the PT obtained from embodiment 1 after the PT standard substance after 100ul dilution and 100 μ l dilution.Put into 37 DEG C after being added a cover by 96 porocyte culture plates again, cultivate 48 hours in 5% carbonic acid gas incubator.After cultivation terminates, take out 96 porocyte culture plates, discard liquid in hole from incubator, add 150 μ l dehydrated alcohols in every hole, room temperature is placed and is fixed for 5 minutes, then discards stationary liquid, and every hole adds 150 μ lPBS, places 1 minute, discards PBS.The rear every hole of end adds 100 μ l Viola crystallina dye liquors and dyes, and after 5 minutes, rinses out dye liquor gently with tap water, is inverted in by cell plate on thieving paper, suck dry moisture.96 orifice plates are placed in observation of cell bunch collection situation under inverted microscope.
The test cell line of PT standard substance is shown in Fig. 6, and the test cell line of the PT obtained in embodiment 1 is shown in Fig. 7, and cell negative control experiments is shown in Fig. 8.Result shows that the PT that obtains in embodiment 1 and standard substance and negative control have obvious difference, and Chinese hamster ovary celI occurs that obvious bunch collects phenomenon.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a separation purification method for pertussis antigen, is characterized in that, comprises the following steps:
(1) diatomite is loaded in chromatography column;
(2) bordetella pertussis fermented liquid centrifugal after get supernatant liquor, be the 5%-10% of original volume by supernatant liquor ultrafiltration and concentration, and adjust conductance;
(3) concentrated supernatant liquor loading, priority PB buffered soln, containing the PB buffered soln of TritonX-100, PB buffered soln chromatography column to be balanced and the wash-out of foreign protein, again with the PB buffered soln elution chromatography post containing 0.15-0.25MNaCl, obtain elutriant 1;
(4) with the PB buffered soln containing 0.5-1.0MNaCl, wash-out is carried out to chromatography column and obtain elutriant 2;
(5) elutriant 1 is through hydroxyapatite chromatography post, and collection penetrates liquid and is Toxins, pertussis;
(6) elutriant 2 is after the absorption of hydroxyapatite chromatography post, and first carry out wash-out containing the PB solution of NaCl to chromatography column with the non-associated proteins of PB eluant solution, again use, the elutriant of collection is Whooping cough filamentous hemagglutinin.
2. the method for claim 1, is characterized in that, diatomite particle diameter is 40-300 μm.
3. the method for claim 1, is characterized in that, after step (1) diatomite dress post, rinses chromatography column with the PB buffered soln of pH7.2-8.0,10-50mmol/L.
4. the method for claim 1, is characterized in that, step (2) fermented liquid centrifugal condition is 3500-7000rpm, and centrifugation time is 30-60min.
5. the method for claim 1, is characterized in that, step (2) adjustment electric conductivity value is 2-8ms/cm.
6. the method for claim 1, it is characterized in that, in step (3), supernatant liquor loading volume is 5-10 times of column volume, successively adopting 10-50mmol/LPB buffered soln to rinse chromatography column to OD value is 0-0.05, PB buffered soln containing 0.05%TritonX-100,10-50mmol/L rinses 5-10 column volume, it is 0-0.05 that the PB buffered soln of 10-50mmol/L rinses chromatography column to OD value, then adopts the PB buffered soln wash-out of NaCl, 10-50mmol/L containing 0.15-0.25M to obtain elutriant 1; Aforementioned PB buffered soln pH is pH7.2-8.0.
7. the method for claim 1, is characterized in that, step (4) rinses chromatography column with the 10-50mmol/LPB buffered soln containing 0.5-1.0MNaCl, pH7.2-8.0, and wash-out obtains elutriant 2.
8. the method as described in as arbitrary in claim 1-7, it is characterized in that, step (6) adopts pH7.2-8.0,10-50mmol/LPB to rinse chromatography column 5-10 column volume, adopt the 0.2-0.5MPB buffered soln elution chromatography post of 0.5-1.0MNaCl, pH7.2-8.0 to be 0-0.05 to OD value again, the elutriant of collection is Whooping cough filamentous hemagglutinin.
9. the application of the arbitrary described method of claim 1-8 in vaccine preparation.
10. apply as claimed in claim 9, it is characterized in that, described vaccine is pertussis vaccine or the vaccine containing pertussis antigen composition.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2021517154A (en) * | 2018-03-27 | 2021-07-15 | グリーン・クロス・コーポレイションＧｒｅｅｎ Ｃｒｏｓｓ Ｃｏｒｐ． | Method for Obtaining Bordetella Pertussis-Derived Protein, Including Affinity Chromatography Step |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1504482A (en) * | 2002-12-03 | 2004-06-16 | 北京优力凯生物技术有限责任公司 | Separation and purification process for acellular whooping cough antigen albumen |
| CN104311647A (en) * | 2014-09-30 | 2015-01-28 | 成都欧林生物科技股份有限公司 | Method for separating and purifying whooping cough toxins and filamentous hemagglutinin |
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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1504482A (en) * | 2002-12-03 | 2004-06-16 | 北京优力凯生物技术有限责任公司 | Separation and purification process for acellular whooping cough antigen albumen |
| CN104311647A (en) * | 2014-09-30 | 2015-01-28 | 成都欧林生物科技股份有限公司 | Method for separating and purifying whooping cough toxins and filamentous hemagglutinin |
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| Title |
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| LARRY U.L.TAN等: "A novel process for preparing an acellular pertussis vaccine composed of non-pyrogenic toxoids of pertussis toxin and filamentous hemagglutinin", 《MOLECULAR IMMUNOLOGY》 * |
| 何金环等: "《生物化学实验技术》", 31 October 2014, 中国轻工业出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021517154A (en) * | 2018-03-27 | 2021-07-15 | グリーン・クロス・コーポレイションＧｒｅｅｎ Ｃｒｏｓｓ Ｃｏｒｐ． | Method for Obtaining Bordetella Pertussis-Derived Protein, Including Affinity Chromatography Step |
| JP7031814B2 (en) | 2018-03-27 | 2022-03-08 | グリーン・クロス・コーポレイション | Methods for Obtaining Bordetella Pertussis-Derived Proteins, Including Affinity Chromatography Steps |
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Application publication date: 20151223 |