CN103833865B - A kind of preparation method of streptococcus pneumoniae capsular polysaccharide - Google Patents
A kind of preparation method of streptococcus pneumoniae capsular polysaccharide Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of streptococcus pneumoniae capsular polysaccharide, the method is comprised the following steps：First, the culture two of streptococcus pneumonia, the inactivation three, extraction four of S. pneumoniae capsular Thick many candies, S. pneumoniae capsular Thick many candies it is refined.
Description
Technical field：
The present invention relates to the preparation of vaccine, more particularly to a kind of preparation method of streptococcus pneumoniae capsular polysaccharide.
Background technology：
Streptococcus pneumonia（streptococcus pneumoniae）Also known as Diplococcus pneumopniae, belong to streptococcus.Pneumonia
Streptococcus can cause various diseases, mainly there is pneumonia, meningitis, bacteremia, tympanitis etc..The bacterium is conditioned pathogen, normally
Carrier state can be formed in the oral cavity of people and nasopharyngeal cavity, disease is caused when Abwehrkraft des Koepers declines, it is especially old
The crowd of people, less than 2 years old children and immune deficiency or hypoimmunity.
Conservative estimation streptococcus pneumonia causes more than 1,000,000 death of child below 5 years old every year in developing country, and extremely
About 20%~25% is the age group in number of dying, or even effectively prevents microorganism infection theory in those willing receiving
Developed country, because morbidity and mortality are also necessary being caused by pneumococcal disease.For example, the U.S. annual about 500
000 streptococcus pneumonia property pneumonia, 50 000 bacteremia, 3 000 streptococcus pneumonia meningitis, they cause 40 000 altogether
People is dead.Streptococcus pneumonia is also the popular unique pathogenic bacteria of tympanitis, and the U.S. has 2 400 ten thousand people to see pediatrician every year,
And use more antibiotic than animal infectious diease.Therefore, tympanitis is infected to the Health Expenditure of developed country with other
Disease estimates that annual expenditure will be more than 5,000,000,000 dollars compared to very big impact is caused.
Streptococcus pneumonia, is gram-positive bacteria.One layer of capsular polysaccharide of its surface attachment, shields to thalline, is
Main virulence factor.Composition difference according to the pod membrane, has identified about 90 different Pneumococcus serotypes, wherein only
Having about 20 several can cause human diseases.Scientist respectively carries the capsular polysaccharide of the pneumococcus of this 20 several different serotypes
Take out, be made vaccine for prevent by the microbial various diseases of pneumonia streptococcus.At present, adult pneumonia vaccine is 23 valency pneumonia
Polysaccharide vaccine, type include 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F,
20th, 22F, 23F and 33F type, 85% ~ 95% epidemic link can be covered in country variant and area.Capsular polysaccharide be T cell it is non-according to
Rely property antigen, less than 2 years old children are immature due to immune system, and polysaccharide vaccine can only produce limited immune response, and
There is no immunological memory to react.Capsular polysaccharide is combined with carrier protein（I.e. so-called polysaccharide-protein combined vaccine）Polysaccharide can be made
Antigen changes into T cell dependence antigen by T cell independent antigen, effectively excites less than the 2 years old immune response of children, and
Immunological memory is formed, the main of the pneumonia polysaccharide-protein combined vaccine on the whole world there are several products such as 7 valencys, 10 valencys, 13 valencys at present
Product.However, which kind of pneumovax product, it is the first step to prepare various other pneumonia polysaccharide.
Pneumococcal capsular polysaccharide is the active principle of pneumovax, and existing preparation method is a lot, but laboratory method compared with
Many, it is difficult to be applied to industrialization, the present invention establishes a kind of pneumonia streptococcus fungus fermentation culturing method and a kind of streptococcus pneumonia pod
Film polysaccharide（Pn-Ps）The new industrial process of preparation.It is characterized in that：1. whole Polyose extraction process it is main with conventional physics,
Chemical Decomposition method, it is to avoid extracted using the organic reagent being commonly used in conventional article（Such as cold phenol method, phenol-chloroform method etc.）
Or the method for the impurity in ferment treatment Polysaccharide removing sample, farthest reduce the injury and environmental pollution to operator；
2. whole extraction process is simple, be easy to grasp；3. the purity of polysaccharide for preparing is high.
The content of the invention：
The present invention provides a kind of fermented and cultured new technology of streptococcus pneumonia, and pod membrane is more in can making nutrient solution using the technique
The yield of sugar meets the demand of production of vaccine enough.In addition, the present invention also provides a kind of extraction of streptococcus pneumoniae capsular polysaccharide
Technique, the nutrient solution of inactivation is through the combination techniques such as centrifugation, UF membrane, segmentation alcohol precipitation, sieve chromatography from culture of streptococcus pneumonia
Pn-Ps is extracted and purified in thing, and whole capsular polysaccharide extraction process avoids being extracted using organic reagent or ferment treatment goes the removal of impurity
Method, and the chemical composition of prepared Pn-Ps meets the requirement of European Pharmacopoeia 5.0 editions, impurity protein, the content point of nucleic acid
Not below 1%.
The preparation method of streptococcus pneumoniae capsular polysaccharide of the invention, by following steps：
First, the culture of streptococcus pneumonia
2nd, inactivate
3rd, the extraction of S. pneumoniae capsular Thick many candies
4th, S. pneumoniae capsular Thick many candies is refined.
Wherein, the culture of the streptococcus pneumonia, including, using sheep blood plate recovery streptococcus pneumonia；Prepare pneumonia chain
Coccus seed liquor；Seed liquor is inoculated into fermentation tank and obtains the nutrient solution containing Pn-Ps through culture.
Wherein, the inactivation, including thalline living is inactivated using conventional method, such as using inactivator to nutrient solution
In streptococcus pneumonia inactivated.
Wherein, the extraction of the S. pneumoniae capsular Thick many candies, including, zymotic fluid passes through continuous stream or cup type centrifugation
After machine centrifugation, using the further clarifying treatment of the modes such as doughnut or depth filter；Used by the zymotic fluid after clarification
100kDa milipore filter bags are concentrated by ultrafiltration；Concentrate redissolves after two sections of alcohol precipitations to precipitation.
Wherein, S. pneumoniae capsular Thick many candies is refined, including, the solution after redissolution carries out essence using sieve chromatography
It is pure；The sample solution of collection is using 30kDa milipore filter bag desalinations and concentrates, and freeze-drying obtains refined polysaccharide.
Wherein, the streptococcus pneumonia bacterial type being applicable include 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F,
14th, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F type（Bacterium source is in ATCC）.
Wherein, the culture of the streptococcus pneumonia, step preferably is：
After being inoculated with fermentation tank, pH controls are used from hair in 6.5-8.0, rotating speed 50-200r/min, culture incipient stage
Fermentation tank bottom leads to compressed gas, and dissolved oxygen amount is controlled in 1-10%, when zymotic fluid bacterium is dense（OD600nm）When reaching 0.5-1, change logical
Gas mode is to lead to compressed gas from the top of fermentation tank, and dissolved oxygen is controlled in 0.01-2%, when zymotic fluid bacterium is dense reaches 2-2.5, is fitted
Amount supplementary carbon source, thalline is inactivated after continuing culture to the plateau of thalli growth.
Particularly preferred step is：
After being inoculated with stirred-tank fermenter, PH is controlled in 7-7.5, rotating speed 70-150r/min, rotating speed culture incipient stage
Lead to compressed gas using from fermenter base, dissolved oxygen amount is controlled in 3-7%, when zymotic fluid bacterium is dense reaches 0.5-1, change ventilation
Mode is to lead to compressed gas from the top of fermentation tank, and dissolved oxygen is controlled in 0.1-1.0%, when zymotic fluid bacterium is dense reaches 2-2.5, is fitted
Amount supplementary carbon source, thalline is inactivated after continuing culture to the plateau of thalli growth.
Most preferred step is：
After being inoculated with stirred-tank fermenter, 37 DEG C ± 2 DEG C of cultivation temperature, pH maintains 7.2 ± 0.2, rotating speed 100r/
Min, the rotating speed culture incipient stage is used from fermenter base leads to compressed gas, and dissolved oxygen amount is controlled 5%, reached when zymotic fluid bacterium is dense
During to 0.5-1, it is to lead to compressed gas from the top of fermentation tank to change ventilating mode, and dissolved oxygen is controlled 0.2%, when zymotic fluid bacterium is dense
When reaching 2-2.5, appropriate supplementary carbon source inactivates thalline after continuing culture to the plateau of thalli growth.
In above step, described is aseptic compressed air to the gas being passed through in fermentation tank, or oxygen and nitrogen
Or the combination gas of carbon dioxide.
Wherein, in the extraction of the S. pneumoniae capsular Thick many candies, step preferably is：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, the small molecule composition in removal thalline, bacterial chip and nutrient solution,
Obtain concentrate；
B, concentrate are slowly added to ethanol to after final concentration of 15-35%, and 2-8 DEG C stands overnight；
C, gained stand liquid centrifugation removal precipitation, and supernatant continues plus ethanol is to after final concentration of 60-90%, and 2-8 DEG C stood
Night；
D, gained stand liquid and precipitation are collected by centrifugation, after precipitation water or NaCl solution are redissolved, centrifugation removal precipitation, on gained
Raw sugar solution is clearly；
Particularly preferred step is：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, removal thalline, bacterial chip, particulate matter and small in nutrient solution
Molecular chaperones, obtain concentrate；
B, concentrate are slowly added to ethanol to after final concentration of 20-30%, and 2-8 DEG C stands overnight；
C, gained stand liquid centrifugation removal precipitation, and supernatant continues plus ethanol is to after final concentration of 75-85%, and 2-8 DEG C stood
Night；
D, gained stand liquid and precipitation are collected by centrifugation, after precipitation water or NaCl solution are redissolved, centrifugation removal precipitation, on gained
Raw sugar solution is clearly；
Most preferred step is：
A, culture of streptococcus pneumonia liquid after the thalline of inactivation is taken, it is broken using 15000g centrifugation 30min removal thalline and thalline
Piece, supernatant further removes the particulate matter of remnants by the way of micro-filtration or in-depth filtration；Solution after clarification is used
The ultrafiltration system of 100kDa is concentrated and removes the small molecule composition in nutrient solution, final to obtain concentrate；
B, concentrate are slowly added to ethanol to after final concentration of 25%, and 2-8 DEG C stands overnight；
C, gained stand liquid centrifugation removal precipitation, and supernatant continues plus ethanol is to after final concentration of 80%, and 2-8 DEG C stands overnight；
D, gained stand liquid 15000g centrifugations 30min and collect precipitation, and precipitation is redissolved using 0.2M NaCl solutions, redissolves liquid
15000g, centrifugation 30min removals precipitate to obtain supernatant, as Thick many candies solution.
Wherein, the S. pneumoniae capsular Thick many candies is refined, and step preferably is：
Gained raw sugar solution, is separated, according to the pneumonia polysaccharide of each type in European Pharmacopoeia using molecular sieve exclusion chromatography
Standard collection macromolecular polysaccharide in 5.0 editions；Polysaccharide after separation uses 30kDa milipore filter bag desalinations, then it is freeze-dried after
Obtain final product streptococcus pneumoniae capsular polysaccharide.
Particularly preferred step is：
To the refined use molecular sieve exclusion chromatography of Thick many candies solution, filler used by molecular sieve exclusion chromatography is optional：
Sepharose CL-2B, Sepharose CL-4B, Sephacryl S-200, Sepharose 4 Fast Flow or
Superdex 200；According to standard collection macromolecular polysaccharide of the pneumonia polysaccharide of each type in European Pharmacopoeia 5.0 editions；Separate
Polysaccharide afterwards use 30kDa milipore filter bag desalinations, then it is freeze-dried after obtain final product streptococcus pneumoniae capsular polysaccharide.
Streptococcus pneumoniae capsular polysaccharide to being prepared to top method has carried out biochemistry detection, as a result as follows：
Biochemical detection methods：Protein content is determined using lowry methods, and nucleic acid content uses determined by ultraviolet spectrophotometry,
Nitrogen content uses Kjeldahl nitrogen determination, phosphorus content to be determined using ammonium molybdate method, and glucuronic acid content uses carbazole-ethanol solution method
Determine, amido hexose content uses diaminobenzene formaldehyde determination of color, methylpentose content is surveyed using cysteine development process
Fixed, O- acetyl contents are determined using alkaline hydroxylamine hydrochloride-tri-chlorination iron processes.The refined unit price polysaccharide biochemistry detection knot of part type
Fruit see the table below：
The univalent polysaccharide biochemistry detection result in part
As can be seen from the above table, the other streptococcus pneumoniae capsular polysaccharide of the different shaped of preparation, each biochemical detection indexes are equal
Meet 5.0 editions requirements of European Pharmacopoeia, wherein impurity（Albumen, nucleic acid）Content is 1% or less than 1%.
The carrying out of exquisite purity of polysaccharide is detected using molecular sieve exclusion chromatography, the application is not as space is limited,
Purity detecting result to all polysaccharide is summarized,
Specific detection method is as follows：
1st, sample ID：Pn19F-Ps
Filler：Sepharose 4 Fast Flow
Chromatographic column specification：xk 50/100
Post bed parameter：V0=562, Vt=1678
Produce higher absorption peak is：206nm absorbances；It is shallower：280nm absorbances
Relative molecular weight kd=0.12 at peak point
Be can be seen that from accompanying drawing 2 in the case of having higher absorption peak under 206nm, absorption value under 280nm（Main generation
Table protein peak）It is very low, it is seen that the polysaccharide of extraction has purity higher.
2nd, sample ID：Pn9V-Ps
Filler：Sepharose 4 Fast Flow
Chromatographic column specification：xk 50/100
Post bed parameter：V0=562, Vt=1678
Produce higher absorption peak is：206nm absorbances；It is shallower：Relative molecular weight kd=at 280nm absorbances peak point
0.076
Be can be seen that from accompanying drawing 3 in the case of having higher absorption peak under 206nm, absorption value under 280nm（Main generation
Table protein peak）It is very low, it is seen that the polysaccharide of extraction has purity higher.
3rd, sample ID：Pn5-Ps
Filler：Sepharose 4 Fast Flow
Chromatographic column specification：xk 50/100
Post bed parameter：V0=562, Vt=1678
Produce higher absorption peak is：206nm absorbances；It is shallower：Relative molecular weight kd=at 280nm absorbances peak point
0.33
Be can be seen that from accompanying drawing 4 in the case of having higher absorption peak under 206nm, absorption value under 280nm（Main generation
Table protein peak）It is very low, it is seen that the polysaccharide of extraction has purity higher.
The beneficial effect of herein described preparation method：
The 1st, molecular sieve exclusion chromatography is used for the extraction of streptococcus pneumoniae capsular polysaccharide first, it can to efficiently separate many
Sugar, goes the removal of impurity, makes the content of impurity protein in end-product, nucleic acid respectively below 1%；
2nd, whole capsular polysaccharide extracting method avoids using organic reagent（Such as phenol, chloroform）Deimpurity side is gone in extracting
Method, it is to avoid injury and pollution that organic reagent is caused to operator and environment；
3rd, whole capsular polysaccharide extracting method avoids going deimpurity method using ferment treatment, while reduces cost not
The impurity new with introducing is worried（Zymoprotein is in itself）；
4th, report the method being combined using two kinds of different ventilating modes first in incubation, training can either be ensured
The fast-growth of the initial stage of supporting thalline, can promote the generation of middle and later periods polysaccharide again, and polysaccharide yield disclosure satisfy that industrialization production
It is required that.
Brief description of the drawings：
Fig. 1, part type streptococcus pneumonia growth curve
Fig. 2, refined polysaccharide（Pn19F-Ps）Molecular sieve exclusion chromatography distribution situation
Fig. 3, refined polysaccharide（Pn9V-Ps）Molecular sieve exclusion chromatography distribution situation
Fig. 4, refined polysaccharide（Pn5-Ps）Molecular sieve exclusion chromatography distribution situation
Specific embodiment：
Embodiment 1：The culture of streptococcus pneumonia 19F and the extraction of Pn19F-Ps
1. the culture of streptococcus pneumonia 19F
Strain comes from American Type Culture Collecti（ATCC 6319）.
The conventional culture methods of streptococcus pneumonia have been that fluid nutrient medium well known in the art, usable includes：TSB、
CY, BHI, Hoeprich ' s culture mediums and its improved formulations etc..
Streptococcus pneumonia 19F types work seed is taken from -70 DEG C of refrigerators, is inoculated in containing 10% de- fiber sheep blood nutrient agar
On flat board, 37 DEG C ± 1 DEG C, 5%CO218-24h is cultivated in incubator.Checking contamination-free.
Above-mentioned culture is scraped, several 1L are seeded in（Culture medium loading amount 500ml）In triangular flask, in 37 DEG C ± 2 DEG C, 5%
CO214-20h is cultivated in incubator, cell density is monitored during this（600nm）.When cell density reaches 2.0-2.5,
Occurred using plate streaking and microscopy method validation contamination-free.
The above-mentioned cultures of 1.5L are inoculated into 50L fermentation tanks（Loading amount 30L）In, condition of culture：37 DEG C ± 2 of cultivation temperature
DEG C, pH maintains 7.2 ± 0.2, rotating speed 100r/min.After inoculation, the culture incipient stage uses empty from the logical compression of fermenter base
The ventilating mode of gas, dissolved oxygen amount is controlled in 1-10%, preferably 3-7%, more excellent 5%.Incubation detection bacterium is dense（OD600nm）.
When bacterium is dense reaches 0.5-1, ventilating mode is made into lead to compressed air from the top of fermentation tank（Ventilate on surface）Ventilating mode,
Dissolved oxygen amount is controlled in 0-2%, preferably 0.1-1%, more excellent 0.2%.2-2.5 is reached when zymotic fluid bacterium is dense, 600ml 50% is supplemented
Glucose, continue cultivate to thalli growth plateau, add final concentration 0.5% phenol inactivation thalline.Subsequently into pod membrane
The polysaccharide extracting process stage.
2. the extraction of streptococcus pneumonia 19F capsular polysaccharides
The above-mentioned nutrient solution for having inactivated, using 15000g centrifugation 30min removal thalline and bacterial chip, supernatant is further
The particulate matter of remnants is removed by the way of micro-filtration or in-depth filtration.Solution after clarification uses the ultrafiltration system of 100kDa
The small molecule composition in nutrient solution is concentrated and removes, it is final to obtain concentrate about 3L.
Substep alcohol precipitation：Above-mentioned concentrate is slowly added to precooling absolute ethyl alcohol while stirring, is added per 100ml concentrates
33.3ml absolute ethyl alcohols, 4 DEG C stand overnight after fully mixing.15000g centrifugation 30min removal precipitations, supernatant continues while stirring
Ethanol to final concentration 80% slowly is added, 4 DEG C stand overnight after fully mixing.15000g centrifugations 30min collects precipitation, and precipitation is adopted
Redissolved with 0.2M NaCl solutions 600ml.15000g centrifugation 30min removals precipitate to obtain supernatant, as Thick many candies solution.
Molecular sieve exclusion chromatography is separated：Above-mentioned supernatant uses molecular sieve exclusion chromatography Sepharose CL-4B（Other molecules
Sieve exclusion chromatography filler Sephacryl S-200, Sepharose 4 Fast Flow, Superdex 200 etc. can also use）
Separate, UV-detector（Wavelength 206nm）Sample peak is monitored with Composition distribution, the sample of relative molecular weight kd≤0.20 is collected
Peak.Polysaccharide sample after separation uses 30kDa milipore filter bag desalinations, then it is freeze-dried after obtain final product refined polysaccharide, refined polysaccharide
Yield is 90-120mg/L zymotic fluids.Refined polysaccharide is stored in less than -20 DEG C refrigerators.Distribution feelings of the refined polysaccharide on chromatographic column
Condition sees appendix two, it can be seen that in the case of having higher absorption peak under 206nm, absorption value under 280nm（Main generation
Table protein peak）It is very low, it is seen that the polysaccharide of extraction has purity higher.
3. refined polysaccharide characterization
（1）Biochemical identification project and method are referring to European Pharmacopoeia 5.0；
（2）Using1H-NMR carries out structure characteristic analysis to refined polysaccharide；
（3）Using Pn19F specific serums（Purchased from Danish National serum research institute）It is using polyacrylamide electrophoresis or reversely electric
Stream immunoelectrophoresis is identified refined polysaccharide；
（4）Using molecular sieve exclusion chromatography（Sepharose CL-4B or Sepharose CL-2B）Determine refined polysaccharide
Relative molecular weight.
Embodiment 2：The culture of streptococcus pneumonia 9V and the extraction of Pn9V-Ps
1. the culture of streptococcus pneumonia 9V
Strain comes from American Type Culture Collecti（ATCC 6309）.
The conventional culture methods of streptococcus pneumonia have been that fluid nutrient medium well known in the art, usable includes：TSB、
CY, BHI, Hoeprich ' s culture mediums and its improved formulations etc..
Streptococcus pneumonia 9V types work seed is taken from -70 DEG C of refrigerators, is inoculated in and is put down containing 10% de- fiber sheep blood nutrient agar
On plate, 36 DEG C ± 1 DEG C, 5%CO218-24h is cultivated in incubator.Checking contamination-free.
Above-mentioned culture is scraped, several 1L are seeded in（Culture medium loading amount 500ml）In triangular flask, in 36 DEG C ± 2 DEG C, 5%
CO214-20h is cultivated in incubator, cell density is monitored during this（OD600nm）.When cell density reaches 2.0-2.5
When, occurred using plate streaking and microscopy method validation contamination-free.
The above-mentioned cultures of 1.5L are inoculated into 50L fermentation tanks（Loading amount 30L）In, condition of culture：36 DEG C ± 2 of cultivation temperature
DEG C, pH maintains 7.0 ± 0.2, rotating speed 80r/min.After inoculation, the culture incipient stage is used from fermenter base leads to compressed air
Ventilating mode, dissolved oxygen amount control in 1-10%, preferably 3-7%, more excellent 4%.Incubation detection bacterium is dense（OD600nm）.When
Bacterium is dense when reaching 0.5-1, and ventilating mode is made into lead to compressed air from the top of fermentation tank（Ventilate on surface）Ventilating mode, it is molten
Oxygen content control is in 0-2%, preferably 0.05-1%, more excellent 0.1%.2-2.5 is reached when zymotic fluid bacterium is dense, supplement 600ml's 50%
Glucose, continues the plateau cultivated to thalli growth, adds the phenol inactivation thalline of final concentration 0.5%.It is many subsequently into pod membrane
The sugared extraction process stage.
2. the extraction of streptococcus pneumonia 9V capsular polysaccharides
The above-mentioned nutrient solution for having inactivated, using 15000g centrifugation 30min removal thalline and bacterial chip, supernatant is further
The particulate matter of remnants is removed by the way of micro-filtration or in-depth filtration.Solution after clarification uses the ultrafiltration system of 100kDa
The small molecule composition in nutrient solution is concentrated and removes, it is final to obtain concentrate about 3L.
Substep alcohol precipitation：Above-mentioned concentrate is slowly added to precooling absolute ethyl alcohol while stirring, is added per 100ml concentrates
33.3ml absolute ethyl alcohols, 4 DEG C stand overnight after fully mixing.15000g centrifugation 30min removal precipitations, supernatant continues while stirring
Ethanol to final concentration 80% slowly is added, 4 DEG C stand overnight after fully mixing.15000g centrifugations 30min collects precipitation, and precipitation is adopted
Redissolved with 0.2M NaCl solutions 600ml.15000g centrifugation 30min removals precipitate to obtain supernatant, as Thick many candies solution.
Molecular sieve exclusion chromatography is separated：Above-mentioned supernatant uses molecular sieve exclusion chromatography Sepharose CL-2B（Other molecules
Sieve exclusion chromatography filler Sephacryl S-200, Sepharose 4 Fast Flow, Superdex 200 etc. can also use）
Separate, UV-detector（Wavelength 206nm）Sample peak is monitored with Composition distribution, the sample of relative molecular weight kd≤0.45 is collected
Peak.Polysaccharide sample after separation uses 30kDa milipore filter bag desalinations, then it is freeze-dried after obtain final product refined polysaccharide, refined polysaccharide
Yield is 100-130mg/L zymotic fluids.Refined polysaccharide is stored in less than -20 DEG C refrigerators.Distribution of the refined polysaccharide on chromatographic column
Situation sees appendix three, it can be seen that in the case of having higher absorption peak under 206nm, absorption value under 280nm（Mainly
Represent protein peak）It is very low, it is seen that the polysaccharide of extraction has purity higher.
3. refined polysaccharide characterization
Referring to the implementation of this partial content in embodiment one, polysaccharide relative molecular weight determines filler and uses
Sepharose CL-2B。
Embodiment 3：The culture of the type of streptococcus pneumonia 5 and the extraction of Pn5-Ps
1. the culture of the type of streptococcus pneumonia 5
Strain comes from American Type Culture Collecti（ATCC 6305）.
The conventional culture methods of streptococcus pneumonia have been that fluid nutrient medium well known in the art, usable includes：TSB、
CY, BHI, Hoeprich ' s culture mediums and its improved formulations etc..
The type of streptococcus pneumonia 5 work seed is taken from -70 DEG C of refrigerators, is inoculated in and is put down containing 10% de- fiber sheep blood nutrient agar
On plate, 37 DEG C ± 2 DEG C, 5%CO218-24h is cultivated in incubator.Checking contamination-free.
Above-mentioned culture is scraped, several 1L are seeded in（Culture medium loading amount 500ml）In triangular flask, in 37 DEG C ± 1 DEG C, 5%
CO214-20h is cultivated in incubator, cell density is monitored during this（600nm）.When cell density reaches 2.0-2.5,
Occurred using plate streaking and microscopy method validation contamination-free.
The above-mentioned cultures of 1.5L are inoculated into 50L fermentation tanks（Loading amount 30L）In, condition of culture：37 DEG C ± 2 of cultivation temperature
DEG C, pH maintains 7.2 ± 0.2, rotating speed 100r/min.After inoculation, the culture incipient stage uses empty from the logical compression of fermenter base
The ventilating mode of gas, dissolved oxygen amount is controlled in 1-10%, preferably 3-7%, more excellent 5%.Incubation detection bacterium is dense（OD600nm）.
When bacterium is dense reaches 0.5-1, ventilating mode is made into lead to compressed air from the top of fermentation tank（Ventilate on surface）Ventilating mode,
Dissolved oxygen amount is controlled in 0-2%, preferably 0.1-1%, more excellent 0.2%.2-2.5 is reached when zymotic fluid bacterium is dense, 600ml 50% is supplemented
Glucose, continue cultivate to thalli growth plateau, add final concentration 0.5% phenol inactivation thalline.Subsequently into pod membrane
The polysaccharide extracting process stage.
2. the extraction of streptococcus pneumonia CP5
The above-mentioned nutrient solution for having inactivated, adds isometric water for injection to reduce the viscosity of zymotic fluid, uses afterwards
15000g centrifugation 30min removal thalline and bacterial chip, supernatant is further removed residual by the way of micro-filtration or in-depth filtration
Remaining particulate matter.Solution after clarification concentrated using the ultrafiltration system of 100kDa and remove the small molecule in nutrient solution into
Point, it is final to obtain concentrate about 3L.
Substep alcohol precipitation：Above-mentioned concentrate is slowly added to precooling absolute ethyl alcohol while stirring, and 25ml is added per 100ml concentrates
Absolute ethyl alcohol, 4 DEG C stand overnight after fully mixing.15000g centrifugation 30min removal precipitations, supernatant continues slow while stirring benefit
Plus ethanol is to final concentration 80%, 4 DEG C stand overnight after fully mixing.15000g centrifugations 30min collects precipitation, and precipitation uses 0.2M
NaCl solution 600ml redissolves.15000g centrifugation 30min removals precipitate to obtain supernatant, as Thick many candies solution.
Molecular sieve exclusion chromatography is separated：Above-mentioned supernatant uses molecular sieve exclusion chromatography Sepharose CL-2B（Other molecules
Sieve exclusion chromatography filler Sephacryl S-200, Sepharose 4 Fast Flow, Superdex 200 etc. can also use）
Separate, UV-detector（Wavelength 206nm）Sample peak is monitored with Composition distribution, the sample of relative molecular weight kd≤0.60 is collected
Peak.Polysaccharide sample after separation uses 30kDa milipore filter bag desalinations, then it is freeze-dried after obtain final product refined polysaccharide, refined polysaccharide
Yield is 70-100mg/L zymotic fluids.Refined polysaccharide is stored in less than -20 DEG C refrigerators.Distribution feelings of the refined polysaccharide on chromatographic column
Condition sees appendix four, it can be seen that in the case of having higher absorption peak under 206nm, absorption value under 280nm（Main generation
Table protein peak）It is very low, it is seen that the polysaccharide of extraction has purity higher.
3. refined polysaccharide characterization
Referring to the implementation of this partial content in embodiment one, polysaccharide relative molecular weight determines filler and uses
Sepharose CL-4B。
Embodiment 4,6B type streptococcus pneumonias
The culture of streptococcus pneumonia, method is as follows：
After stirred-tank fermenter inoculation, pH controls are used from fermentation tank bottom in 6.5, rotating speed 50r/min, culture incipient stage
Portion leads to compressed gas, and dissolved oxygen amount is controlled 1%, when zymotic fluid bacterium is dense（OD600nm）When reaching 0.5, it is from hair to change ventilating mode
The logical compressed gas in the top of fermentation tank, dissolved oxygen is controlled 0.01%, and when zymotic fluid bacterium is dense reaches 2, appropriate supplementary carbon source continues to train
Support and inactivate thalline to the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, the small molecule composition in removal thalline, bacterial chip and nutrient solution,
Obtain concentrate；B, concentrate are slowly added to ethanol to after final concentration of 15%, and 2 DEG C stand overnight；C, gained stand liquid centrifugation and go
Except precipitation, supernatant continues plus ethanol is to after final concentration of 60%, and 2 DEG C stand overnight；D, gained stand liquid and precipitation are collected by centrifugation, and sink
After shallow lake water or NaCl solution are redissolved, centrifugation removal precipitation, gained supernatant is raw sugar solution；
S. pneumoniae capsular Thick many candies it is refined, method is as follows：
Gained raw sugar solution, is separated using the molecular sieve exclusion chromatographies of Superdex 200, and the pneumonia according to each type is more
Standard collection macromolecular polysaccharide of the sugar in European Pharmacopoeia 5.0 editions；Polysaccharide after separation uses 30kDa milipore filter bag desalinations, then
Streptococcus pneumoniae capsular polysaccharide is obtained final product after freeze-dried.
The type streptococcus pneumonia of embodiment 5,8
The culture of streptococcus pneumonia, method is as follows：
After stirred-tank fermenter inoculation, pH controls are used from fermentation tank in 8.0, rotating speed 200r/min, culture incipient stage
Bottom leads to compressed gas, and dissolved oxygen amount is controlled 10%, when zymotic fluid bacterium is dense（OD600nm）When reaching 1, change ventilating mode be from
The logical compressed gas in the top of fermentation tank, dissolved oxygen is controlled 2%, and when zymotic fluid bacterium is dense reaches 2.5, appropriate supplementary carbon source continues to train
Support and inactivate thalline to the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, the small molecule composition in removal thalline, bacterial chip and nutrient solution,
Obtain concentrate；B, concentrate are slowly added to ethanol to after final concentration of 35%, and 8 DEG C stand overnight；C, gained stand liquid centrifugation and go
Except precipitation, supernatant continues plus ethanol is to after final concentration of 90%, and 8 DEG C stand overnight；D, gained stand liquid and precipitation are collected by centrifugation, and sink
After shallow lake water or NaCl solution are redissolved, centrifugation removal precipitation, gained supernatant is raw sugar solution；
S. pneumoniae capsular Thick many candies it is refined, method is as follows：
Gained raw sugar solution, is separated using the molecular sieve exclusion chromatographies of Superdex 200, and the pneumonia according to each type is more
Standard collection macromolecular polysaccharide of the sugar in European Pharmacopoeia 5.0 editions；Polysaccharide after separation uses 30kDa milipore filter bag desalinations, then
Streptococcus pneumoniae capsular polysaccharide is obtained final product after freeze-dried.
The type streptococcus pneumonia of embodiment 6,4
The culture of streptococcus pneumonia, method is as follows：
After stirred-tank fermenter inoculation, pH controls are used from fermentation tank bottom in 7, rotating speed 100r/min, culture incipient stage
Portion leads to compressed gas, and dissolved oxygen amount is controlled 5%, when zymotic fluid bacterium is dense（OD600nm）When reaching 0.75, change ventilating mode be from
The logical compressed gas in the top of fermentation tank, dissolved oxygen is controlled 1%, and when zymotic fluid bacterium is dense reaches 2.1, appropriate supplementary carbon source continues to train
Support and inactivate thalline to the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, removal thalline, bacterial chip, particulate matter is small in nutrient solution
Molecular chaperones, obtain concentrate；B, concentrate are slowly added to ethanol to after final concentration of 20%, and 5 DEG C stand overnight；C, gained stand
Liquid centrifugation removal precipitation, supernatant continues plus ethanol is to after final concentration of 75%, and 5 DEG C stand overnight；D, gained stand liquid and are collected by centrifugation
After precipitation, precipitation water or NaCl solution are redissolved, centrifugation removal precipitation, gained supernatant is raw sugar solution；
S. pneumoniae capsular Thick many candies it is refined, method is as follows：
Gained raw sugar solution, is separated, according to each type using the Fast Flow molecular sieves exclusion chromatographies of Sepharose 4
Standard collection macromolecular polysaccharide of the pneumonia polysaccharide in European Pharmacopoeia 5.0 editions；Polysaccharide after separation uses 30kDa milipore filter bags
Desalination, then it is freeze-dried after obtain final product streptococcus pneumoniae capsular polysaccharide.
The type streptococcus pneumonia of embodiment 7,3
The culture of streptococcus pneumonia, method is as follows：
After stirred-tank fermenter inoculation, pH controls are used from fermentation tank in 7.5, rotating speed 150r/min, culture incipient stage
Bottom leads to compressed gas, and dissolved oxygen amount is controlled 8%, when zymotic fluid bacterium is dense（OD600nm）When reaching 0.6, change ventilating mode be from
The logical compressed gas in the top of fermentation tank, dissolved oxygen is controlled 0.05%, when zymotic fluid bacterium is dense reaches 2.2, appropriate supplementary carbon source, after
Thalline is inactivated after continuous culture to the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, removal thalline, bacterial chip, particulate matter is small in nutrient solution
Molecular chaperones, obtain concentrate；B, concentrate are slowly added to ethanol to after final concentration of 30%, and 8 DEG C stand overnight；C, gained are quiet
Liquid centrifugation removal precipitation is put, supernatant continues plus ethanol is to after final concentration of 85%, and 8 DEG C stand overnight；D, gained stand liquid centrifugation
Precipitation is collected, after precipitation water or NaCl solution are redissolved, centrifugation removal precipitation, gained supernatant is raw sugar solution；
S. pneumoniae capsular Thick many candies it is refined, method is as follows：
Gained raw sugar solution, is separated, according to the pneumonia of each type using Sephacryl S-200 molecular sieves exclusion chromatography
Standard collection macromolecular polysaccharide of the polysaccharide in European Pharmacopoeia 5.0 editions；Polysaccharide after separation uses 30kDa milipore filter bag desalinations,
Streptococcus pneumoniae capsular polysaccharide is obtained final product after freeze-dried again.
The type streptococcus pneumonia of embodiment 8,2
The culture of streptococcus pneumonia, method is as follows：
After stirred-tank fermenter inoculation, 7, rotating speed 70r/min, the rotating speed culture incipient stage is used from fermentation tank for PH controls
Bottom leads to compressed gas, and dissolved oxygen amount is controlled 3%, and when zymotic fluid bacterium is dense reaches 0.5, it is from fermentation tank to change ventilating mode
Top leads to compressed gas, and dissolved oxygen is controlled 0.1%, and when zymotic fluid bacterium is dense reaches 2, appropriate supplementary carbon source continues to cultivate to thalline
Thalline is inactivated after the plateau of growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, the small molecule composition in removal thalline, bacterial chip and nutrient solution,
Obtain concentrate；B, concentrate are slowly added to ethanol to after final concentration of 15%, and 2 DEG C stand overnight；C, gained stand liquid centrifugation and go
Except precipitation, supernatant continues plus ethanol is to after final concentration of 60%, and 2 DEG C stand overnight；D, gained stand liquid and precipitation are collected by centrifugation, and sink
After shallow lake water or NaCl solution are redissolved, centrifugation removal precipitation, gained supernatant is raw sugar solution；
S. pneumoniae capsular Thick many candies it is refined, method is as follows：
Gained raw sugar solution, is separated, according to the pneumonia of each type using Sepharose CL-4B molecular sieves exclusion chromatography
Standard collection macromolecular polysaccharide of the polysaccharide in European Pharmacopoeia 5.0 editions；Polysaccharide after separation uses 30kDa milipore filter bag desalinations,
Streptococcus pneumoniae capsular polysaccharide is obtained final product after freeze-dried again.
The type streptococcus pneumonia of embodiment 9,1
The culture of streptococcus pneumonia, method is as follows：
After stirred-tank fermenter inoculation, 7.5, rotating speed 150r/min, the rotating speed culture incipient stage is used from hair for PH controls
Fermentation tank bottom leads to compressed gas, and dissolved oxygen amount is controlled 7%, and when zymotic fluid bacterium is dense reaches 1, it is from fermentation tank to change ventilating mode
The logical compressed gas in top, dissolved oxygen is controlled 1.0%, and when zymotic fluid bacterium is dense reaches 2.5, appropriate supplementary carbon source continues to cultivate extremely
Thalline is inactivated after the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, the small molecule composition in removal thalline, bacterial chip and nutrient solution,
Obtain concentrate；B, concentrate are slowly added to ethanol to after final concentration of 35%, and 8 DEG C stand overnight；C, gained stand liquid centrifugation and go
Except precipitation, supernatant continues plus ethanol is to after final concentration of 90%, and 8 DEG C stand overnight；D, gained stand liquid and precipitation are collected by centrifugation, and sink
After shallow lake water or NaCl solution are redissolved, centrifugation removal precipitation, gained supernatant is raw sugar solution；
S. pneumoniae capsular Thick many candies it is refined, method is as follows：
Gained raw sugar solution, is separated, according to the pneumonia of each type using Sepharose CL-2B molecular sieves exclusion chromatography
Standard collection macromolecular polysaccharide of the polysaccharide in European Pharmacopoeia 5.0 editions；Polysaccharide after separation uses 30kDa milipore filter bag desalinations,
Streptococcus pneumoniae capsular polysaccharide is obtained final product after freeze-dried again.
The preparation of other kinds of streptococcus pneumoniae capsular polysaccharide is such as： 7F、8、9N、9V、10A、11A、12F、14、15B、
17F, 18C, 19A, 19F, 20,22F, 23F and 33F type, preparation method are same as Example 1.
Claims (8)
1. a kind of preparation method of streptococcus pneumoniae capsular polysaccharide, the method is comprised the following steps, and the culture of streptococcus pneumonia is gone out
It is living, the extraction of S. pneumoniae capsular Thick many candies, S. pneumoniae capsular Thick many candies it is refined, it is characterised in that
The culture of the streptococcus pneumonia, method is as follows：
After stirred-tank fermenter inoculation, pH controls are used from fermentation in 6.5-8.0, rotating speed 50-200r/min, culture incipient stage
Pot bottom leads to compressed gas, and dissolved oxygen amount is controlled in 1-10%, when zymotic fluid bacterium is dense reaches 0.5-1, change ventilating mode be from
The logical compressed gas in the top of fermentation tank, dissolved oxygen is controlled in 0.01-2%, when zymotic fluid bacterium is dense reaches 2-2.5, carbon is supplemented in right amount
Source, thalline is inactivated after continuing culture to the plateau of thalli growth；
The extraction of the S. pneumoniae capsular Thick many candies, method is as follows：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, the small molecule composition in removal thalline, bacterial chip and nutrient solution obtains dense
Contracting liquid；
B, concentrate are slowly added to ethanol to after final concentration of 15-35%, and 2-8 DEG C stands overnight；
C, gained stand liquid centrifugation removal precipitation, and supernatant continues plus ethanol is to after final concentration of 60-90%, and 2-8 DEG C stands overnight；
D, gained stand liquid and precipitation are collected by centrifugation, and after precipitation water or NaCl solution are redissolved, centrifugation removal is precipitated, and gained supernatant is
It is raw sugar solution；
The S. pneumoniae capsular Thick many candies it is refined, method is as follows：
Gained raw sugar solution, is separated using molecular sieve exclusion chromatography, according to the pneumonia polysaccharide of each type in European Pharmacopoeia 5.0 editions
In standard collection macromolecular polysaccharide；Polysaccharide after separation uses 30kDa milipore filter bag desalinations, then it is freeze-dried after obtain final product lung
Scorching streptococcal capsular polysaccharide.
2. preparation method as described in claim 1, it is characterised in that the extraction of the S. pneumoniae capsular Thick many candies,
Step is as follows：
A, the culture of streptococcus pneumonia liquid after inactivation is taken, removal thalline, bacterial chip, particulate matter and small molecule in nutrient solution
Composition, obtains concentrate；
B, concentrate are slowly added to ethanol to after final concentration of 20-30%, and 2-8 DEG C stands overnight；
C, gained stand liquid centrifugation removal precipitation, and supernatant continues plus ethanol is to after final concentration of 75-85%, and 2-8 DEG C stands overnight；
D, gained stand liquid and precipitation are collected by centrifugation, and after precipitation water or NaCl solution are redissolved, centrifugation removal is precipitated, and gained supernatant is
It is raw sugar solution.
3. preparation method as described in claim 1, it is characterised in that the extraction of the S. pneumoniae capsular Thick many candies,
Step is as follows：
A, culture of streptococcus pneumonia liquid after the thalline of inactivation is taken, using 15000g centrifugation 30min removal thalline and bacterial chip, on
Clear liquid further removes the particulate matter of remnants by the way of micro-filtration or in-depth filtration；Solution after clarification uses 100kDa
Ultrafiltration system concentrate and remove the small molecule composition in nutrient solution, final concentrate；
B, concentrate are slowly added to ethanol to after final concentration of 25%, and 2-8 DEG C stands overnight；
C, gained stand liquid centrifugation removal precipitation, and supernatant continues plus ethanol is to after final concentration of 80%, and 2-8 DEG C stands overnight；
D, gained stand liquid 15000g centrifugations 30min and collect precipitation, and precipitation is redissolved using 0.2M NaCl solutions, redissolves liquid
15000g, centrifugation 30min removals precipitate to obtain supernatant, as Thick many candies solution.
4. preparation method as described in claim 1, it is characterised in that filler used by molecular sieve exclusion chromatography is selected from：
Sepharose CL-2B, Sepharose CL-4B, Sephacryl S-200, Sepharose 4Fast Flow or
Superdex 200；According to standard collection macromolecular polysaccharide of the pneumonia polysaccharide of each type in European Pharmacopoeia 5.0 editions；Separate
Polysaccharide afterwards use 30kDa milipore filter bag desalinations, then it is freeze-dried after obtain final product streptococcus pneumoniae capsular polysaccharide.
5. preparation method as claimed in claim 1, it is characterised in that the culture of the streptococcus pneumonia, step is as follows：Stirring
After the inoculation of formula fermentation tank, in 7-7.5, rotating speed 70-150r/min, the culture incipient stage is used from the logical pressure of fermenter base for pH controls
Contracting gas, dissolved oxygen amount is controlled in 3-7%, and when zymotic fluid bacterium is dense reaches 0.5-1, it is from the top of fermentation tank to change ventilating mode
Logical compressed gas, dissolved oxygen is controlled in 0.1-1.0%, and when zymotic fluid bacterium is dense reaches 2-2.5, appropriate supplementary carbon source continues to cultivate
Thalline is inactivated after to the plateau of thalli growth.
6. preparation method as claimed in claim 1, it is characterised in that the culture of the streptococcus pneumonia, step is as follows：Stirring
After the inoculation of formula fermentation tank, 37 DEG C ± 2 DEG C of cultivation temperature, pH maintains 7.2 ± 0.2, and rotating speed 100r/min, rotating speed culture starts rank
Duan Caiyong leads to compressed gas from fermenter base, and dissolved oxygen amount is controlled 5%, when zymotic fluid bacterium is dense reaches 0.5-1, changes ventilation
Mode is to lead to compressed gas from the top of fermentation tank, and dissolved oxygen is controlled 0.2%, appropriate to mend when zymotic fluid bacterium is dense reaches 2-2.5
Carbon source is filled, thalline is inactivated after continuing culture to the plateau of thalli growth.
7. preparation method as claimed in claim 1, it is characterised in that the bacterial type that the cultural method is applicable includes 1,2,3,
4th, 5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F type.
8. preparation method as claimed in claim 1, it is characterised in that to the gas being passed through in fermentation tank be aseptic compression
Air, or oxygen and nitrogen or the combination gas of carbon dioxide.
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| CN101129439A (en) * | 2007-08-01 | 2008-02-27 | 山西医科大学 | Method for extracting codonopsis pilosula polyoses |
| CN101486772A (en) * | 2009-02-13 | 2009-07-22 | 南京师范大学 | Mistletoe polysaccharide, as well as preparation and use thereof |
| CN102617745A (en) * | 2012-03-05 | 2012-08-01 | 广东省微生物研究所 | Preparation method and blood sugar lowering function of Ganoderma lucidum karst polysaccharide F31 |
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| CN101129439A (en) * | 2007-08-01 | 2008-02-27 | 山西医科大学 | Method for extracting codonopsis pilosula polyoses |
| CN101486772A (en) * | 2009-02-13 | 2009-07-22 | 南京师范大学 | Mistletoe polysaccharide, as well as preparation and use thereof |
| CN102617745A (en) * | 2012-03-05 | 2012-08-01 | 广东省微生物研究所 | Preparation method and blood sugar lowering function of Ganoderma lucidum karst polysaccharide F31 |
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| 肺炎链球菌荚膜多糖生产工艺的建立;金永杰等;《2010年中国药学大会暨第十届中国药师周大会论文集》;20110728;1-6 * |
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