CN103833865A - Method for preparing streptococcus pneumoniae capsular polysaccharide - Google Patents
Method for preparing streptococcus pneumoniae capsular polysaccharide Download PDFInfo
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- CN103833865A CN103833865A CN201210490414.8A CN201210490414A CN103833865A CN 103833865 A CN103833865 A CN 103833865A CN 201210490414 A CN201210490414 A CN 201210490414A CN 103833865 A CN103833865 A CN 103833865A
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Images
Abstract
The invention relates to a method for preparing streptococcus pneumoniae capsular polysaccharide. The method comprises the following steps: 1, culturing streptococcus pneumoniae; 2, inactivating; 3, extracting streptococcus pneumoniae capsular polysaccharide; 4, refining streptococcus pneumoniae capsular polysaccharide.
Description
Technical field:
The present invention relates to the preparation of vaccine, particularly a kind of preparation method of streptococcus pneumoniae capsular polysaccharide.
Background technology:
Streptococcus pneumoniae (streptococcus pneumoniae) has another name called pneumococcus, belongs to streptococcus.Streptococcus pneumoniae can cause various diseases, mainly contains pneumonia, meningitis, microbemia, otitis media etc.This bacterium is conditioned pathogen, all can form carrier state in normal people's oral cavity and cavum nasopharyngeum, when Abwehrkraft des Koepers declines, causes disease, especially the crowd of the elderly, children below 2 years old and immune deficiency or hypoimmunity.
Conservative estimation streptococcus pneumoniae causes more than 1,000,000 death of child below 5 years old every year in developing country, and about 20%～25% in death toll, this age group, even effectively prevent the developed country of infected by microbes theory in those willing acceptance, the M & M causing because of pneumonia disease is also necessary being.For example, the annual nearly 500 000 routine streptococcus pneumoniae property pneumonia of the U.S., 50 000 routine microbemia, 3 000 routine streptococcus pneumoniae meningitis, they cause 40 000 people's death altogether.Streptococcus pneumoniae is also unique pathogenic bacterium that otitis media is popular, and the U.S. has 2 400 ten thousand people will see pediatrician every year, and has used more microbiotic than other transmissible diseases.Therefore, otitis media has caused very large impact compared with other transmissible disease to the Health Expenditure of developed country, estimates that annual expenditure will exceed 5,000,000,000 dollars.
Streptococcus pneumoniae is gram-positive microorganism.Its surface attachment one deck capsular polysaccharide, shields to thalline, is main virulence factor.According to the composition difference of this pod membrane, identify approximately 90 different streptococcus pneumoniae serotypes, wherein only having an appointment 20 severally can cause human diseases.Scientist extracts the pneumococcal capsular polysaccharide of these 20 several different serotypes respectively, makes vaccine for preventing by the microbial various diseases of pneumonia streptococcus.At present, adult pneumonia vaccine is 23 valency pneumonia polysaccharide vaccines, type comprises 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F type, can cover 85% ~ 95% popular bacterial strain at country variant and area.Capsular polysaccharide is T cell dependent/non-dependent antigen, and 2 years old following children is due to immune immature, and polysaccharide vaccine can only produce limited immunne response, and there is no immunological memory reaction.Capsular polysaccharide is combined with carrier proteins (being so-called polysaccharide-protein combined vaccine) can make polysaccharide antigen become T cell dependence antigen by T cell dependent/non-dependent Antigenic conversion, effectively excite 2 years old following children's immunne response, and form immunological memory, several products such as 7 valencys that mainly contain of the pneumonia polysaccharide-protein combined vaccine on the whole world, 10 valencys, 13 valencys at present.No matter but which kind of pneumovax product, preparing various other pneumonia polysaccharide is all the first steps.
Pneumococcal capsular polysaccharide is the active principle of pneumovax, existing preparation method is a lot, but laboratory method is more, be difficult to be applied to industrialization, the present invention has set up new industrial process prepared by a kind of pneumonia streptococcus fungus fermentation culturing method and a kind of streptococcus pneumoniae capsular polysaccharide (Pn-Ps).It is characterized in that: 1. whole polysaccharide leaching process is mainly with conventional physics, chemical separation method, avoid adopting the organic reagent extracting (such as cold phenol method, phenol-chloroform method etc.) that often uses in article in the past or enzyme to process the method for the impurity in Polysaccharide removing sample, farthest reduced injury and environmental pollution to operator; 2. whole leaching process simple, be easy to grasp; 3. the purity of polysaccharide of preparation is high.
Summary of the invention:
The invention provides the fermentation culture novel process of a kind of streptococcus pneumoniae, adopt this technique can make the output of capsular polysaccharide in nutrient solution enough meet the demand of production of vaccine.In addition, the present invention also provides a kind of extraction process of streptococcus pneumoniae capsular polysaccharide, the nutrient solution of deactivation extracts and purifying Pn-Ps from culture of streptococcus pneumonia thing through combination techniques such as centrifugal, membrane sepn, segmentation alcohol precipitation, sieve chromatographies, whole capsular polysaccharide leaching process avoids using organic reagent extracting or enzyme processing to go deimpurity method, and the chemical constitution of prepared Pn-Ps meets 5.0 editions requirements of European Pharmacopoeia, and the content of impurity albumen, nucleic acid is respectively below 1%.
The preparation method of streptococcus pneumoniae capsular polysaccharide of the present invention, process following steps:
One, the cultivation of streptococcus pneumoniae
Two, deactivation
Three, the extraction of streptococcus pneumoniae pod membrane Crude polysaccharides
Four, streptococcus pneumoniae pod membrane Crude polysaccharides is refining.
Wherein, the cultivation of described streptococcus pneumoniae, comprises, adopts sheep blood agar recovery streptococcus pneumoniae; Preparation streptococcus pneumoniae seed liquor; Seed liquor is inoculated in fermentor tank and obtains through cultivating the nutrient solution that contains Pn-Ps.
Wherein, described deactivation, comprises and uses ordinary method to carry out deactivation to the thalline of living, as used inactivator to carry out deactivation to the streptococcus pneumoniae in nutrient solution.
Wherein, the extraction of described streptococcus pneumoniae pod membrane Crude polysaccharides, comprises, fermented liquid by Continuous Flow or cup type whizzer centrifugal after, adopt the further clarifying treatment of mode such as tubular fibre or depth filter; Carry out ultrafiltration and concentration through the fermented liquid after clarification with 100kDa ultra-filtration membrane bag; Concentrated solution redissolves to precipitation after two sections of alcohol precipitations.
Wherein, refining of streptococcus pneumoniae pod membrane Crude polysaccharides, comprises, the solution after redissolution adopts sieve chromatography to carry out consummate; The sample solution of collecting adopts 30kDa ultra-filtration membrane bag desalination concentrated, and lyophilize obtains refining polysaccharide.
Wherein, applicable streptococcus pneumoniae bacterial type comprise 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F type (bacterium source is in ATCC).
Wherein, the cultivation of described streptococcus pneumoniae, preferred step is:
After inoculating in fermentor tank, pH is controlled at 6.5-8.0, rotating speed 50-200r/min, cultivates initial stage employing from the logical pressurized gas in fermentor tank bottom, and dissolved oxygen amount is controlled at 1-10%, in the time that fermented liquid bacterium dense (OD600nm) reaches 0.5-1, changing ventilating mode is logical pressurized gas from the top of fermentor tank, and dissolved oxygen is controlled at 0.01-2%, when fermented liquid bacterium is dense while reaching 2-2.5, appropriate supplementary carbon source, continues to be cultured to deactivation thalline after the plateau of thalli growth.
Particularly preferred step is:
After inoculating in stirred-tank fermenter, PH is controlled at 7-7.5, rotating speed 70-150r/min, rotating speed is cultivated initial stage employing from the logical pressurized gas in fermentor tank bottom, and dissolved oxygen amount is controlled at 3-7%, when fermented liquid bacterium is dense while reaching 0.5-1, changing ventilating mode is logical pressurized gas from the top of fermentor tank, and dissolved oxygen is controlled at 0.1-1.0%, when fermented liquid bacterium is dense while reaching 2-2.5, appropriate supplementary carbon source, continues to be cultured to deactivation thalline after the plateau of thalli growth.
Most preferred step is:
After inoculating in stirred-tank fermenter, 37 ℃ ± 2 ℃ of culture temperature, pH maintains 7.2 ± 0.2, rotating speed 100r/min, rotating speed is cultivated initial stage employing from the logical pressurized gas in fermentor tank bottom, dissolved oxygen amount is controlled at 5%, and when fermented liquid bacterium is dense while reaching 0.5-1, changing ventilating mode is the logical pressurized gas in top from fermentor tank, dissolved oxygen is controlled at 0.2%, when fermented liquid bacterium is dense while reaching 2-2.5, appropriate supplementary carbon source, continues to be cultured to deactivation thalline after the plateau of thalli growth.
In above step, described is aseptic pressurized air to the gas passing in fermentor tank, or the composition gas of oxygen and nitrogen or carbonic acid gas.
Wherein, in the extraction of described streptococcus pneumoniae pod membrane Crude polysaccharides, preferred step is:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove the small molecules composition in thalline, bacterial chip and nutrient solution, obtain concentrated solution;
After b, concentrated solution slowly add ethanol to final concentration to be 15-35%, 2-8 ℃ of hold over night;
C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 60-90%, and 2-8 ℃ of hold over night;
D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution;
Particularly preferred step is:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove particulate matter and small molecules composition in thalline, bacterial chip, nutrient solution, obtain concentrated solution;
After b, concentrated solution slowly add ethanol to final concentration to be 20-30%, 2-8 ℃ of hold over night;
C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 75-85%, and 2-8 ℃ of hold over night;
D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution;
Most preferred step is:
A, get culture of streptococcus pneumonia liquid after the thalline of deactivation, adopt the centrifugal 30min of 15000g to remove thalline and bacterial chip, supernatant liquor further adopts the mode of micro-filtration or Depth Filtration to remove remaining particulate matter; Solution after clarification adopts the ultrafiltration system of 100kDa to concentrate and remove the small molecules composition in nutrient solution, the final concentrated solution that obtains;
After b, concentrated solution slowly add ethanol to final concentration to be 25%, 2-8 ℃ of hold over night;
C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 80%, and 2-8 ℃ of hold over night;
D, gained leave standstill the centrifugal 30min collecting precipitation of liquid 15000g, and precipitation adopts 0.2M NaCl solution to redissolve, redissolution liquid 15000g, and centrifugal 30min removes and precipitates to obtain supernatant, is Crude polysaccharides solution.
Wherein, described streptococcus pneumoniae pod membrane Crude polysaccharides refining, preferred step is:
Gained raw sugar solution, adopts molecular sieve exclusion chromatography to separate, and collects macromolecular polysaccharide according to the pneumonia polysaccharide of each type in the standard in European Pharmacopoeia 5.0 editions; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
Particularly preferred step is:
To the refining employing molecular sieve exclusion chromatography of Crude polysaccharides solution, molecular sieve exclusion chromatography filler used is optional: Sepharose CL-2B, Sepharose CL-4B, Sephacryl S-200, Sepharose 4 Fast Flow or Superdex 200; Standard according to the pneumonia polysaccharide of each type in European Pharmacopoeia 5.0 editions is collected macromolecular polysaccharide; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
To having carried out biochemistry detection in order to the standby streptococcus pneumoniae capsular polysaccharide of top legal system, result is as follows:
Biochemistry detection method: protein content adopts lowry method to measure; nucleic acid content adopts determined by ultraviolet spectrophotometry; nitrogen content adopts Kjeldahl nitrogen determination; phosphorus content adopts ammonium molybdate method to measure; glucuronic acid content adopts carbazole-ethanolic soln method to measure; amido hexose content adopts diaminobenzene formaldehyde determination of color, and methylpentose content adopts halfcystine determination of color, and O-acetyl content adopts alkaline oxammonium hydrochloride-tri-chlorination iron processes to measure.The refining unit price polysaccharide biochemistry detection of part type the results are shown in following table:
Part unit price polysaccharide biochemistry detection result
As can be seen from the above table, other streptococcus pneumoniae capsular polysaccharide of the different shaped of preparation, each biochemistry detection index all meets 5.0 editions requirements of European Pharmacopoeia, and wherein impurity (albumen, nucleic acid) content is all below 1% or 1%.
Adopt molecular sieve exclusion chromatography to exquisite purity of polysaccharide detect, the application does not summarize the purity detecting result of all polysaccharide as space is limited,,
Concrete detection method is as follows:
1, sample title: Pn19F-Ps
Filler: Sepharose 4 Fast Flow
Chromatography column specification: xk 50/100
Post bed parameter: V0=562, Vt=1678
What produce higher absorption peak is: 206nm absorbancy; Milder: 280nm absorbancy
The relative molecular weight kd=0.12 of point place, peak
From accompanying drawing 2, can find out, the in the situation that of having higher absorption peak under 206nm, absorption value under 280nm (mainly representing protein peak) is very low, and the visible polysaccharide extracting has higher purity.
2, sample title: Pn9V-Ps
Filler: Sepharose 4 Fast Flow
Chromatography column specification: xk 50/100
Post bed parameter: V0=562, Vt=1678
What produce higher absorption peak is: 206nm absorbancy; Milder: the relative molecular weight kd=0.076 of point place, 280nm absorbancy peak
From accompanying drawing 3, can find out, the in the situation that of having higher absorption peak under 206nm, absorption value under 280nm (mainly representing protein peak) is very low, and the visible polysaccharide extracting has higher purity.
3, sample title: Pn5-Ps
Filler: Sepharose 4 Fast Flow
Chromatography column specification: xk 50/100
Post bed parameter: V0=562, Vt=1678
What produce higher absorption peak is: 206nm absorbancy; Milder: the relative molecular weight kd=0.33 of point place, 280nm absorbancy peak
From accompanying drawing 4, can find out, the in the situation that of having higher absorption peak under 206nm, absorption value under 280nm (mainly representing protein peak) is very low, and the visible polysaccharide extracting has higher purity.
Preparation method's beneficial effect described in the application:
1, the first extraction for streptococcus pneumoniae capsular polysaccharide by molecular sieve exclusion chromatography, it is separating polyose effectively, removes impurity, makes the content of impurity albumen, nucleic acid in end product respectively below 1%;
2, whole capsular polysaccharide extracting method avoids using organic reagent (as phenol, chloroform etc.) extracting to go deimpurity method, injury and the pollution of avoiding organic reagent to cause operator and environment;
3, whole capsular polysaccharide extracting method avoids using enzyme processing to go deimpurity method, does not worry introducing new impurity (zymoprotein itself) in reducing costs;
4, in culturing process, reported first adopts two kinds of methods that different ventilating modes combines, and can either guarantee the Fast Growth of Initial stage of culture thalline, can promote again the generation of middle and later periods polysaccharide, and polysaccharide yield can meet the requirement that industrialization is produced.
Accompanying drawing explanation:
Fig. 1, part type streptococcus pneumoniae growth curve
Fig. 2, refining polysaccharide (Pn19F-Ps) molecular sieve exclusion chromatography distribution situation
Fig. 3, refining polysaccharide (Pn9V-Ps) molecular sieve exclusion chromatography distribution situation
Fig. 4, refining polysaccharide (Pn5-Ps) molecular sieve exclusion chromatography distribution situation
Embodiment:
Embodiment 1: the cultivation of streptococcus pneumoniae 19F and the extraction of Pn19F-Ps
1. the cultivation of streptococcus pneumoniae 19F
Bacterial classification is from American Type Culture Collecti (ATCC 6319).
The cellar culture method of streptococcus pneumoniae has been that this area is in common knowledge, and spendable liquid nutrient medium comprises: TSB, CY, BHI, Hoeprich ' s substratum and improved formulations thereof etc.
From-70 ℃ of refrigerators, get streptococcus pneumoniae 19F type work seed, be inoculated in containing on 10% defiber sheep blood nutrient agar panel 37 ℃ ± 1 ℃, 5%CO
2in incubator, cultivate 18-24h.Checking contamination-free.
Above-mentioned culture is scraped, is seeded in several 1L(substratum loading amount 500ml) in triangular flask, in 37 ℃ ± 2 ℃, 5%CO
2in incubator, cultivate 14-20h, in this process, monitor cell density (600nm).In the time that cell density reaches 2.0-2.5, adopt plate streaking and microscopy method validation contamination-free to occur.
Above-mentioned 1.5L culture is inoculated in 50L fermentor tank (loading amount 30L) to culture condition: 37 ℃ ± 2 ℃ of culture temperature, pH maintains 7.2 ± 0.2, rotating speed 100r/min.After inoculation, cultivate the initial stage to adopt the logical compressed-air actuated ventilating mode from fermentor tank bottom, dissolved oxygen amount is controlled at 1-10%, preferably 3-7%, more excellent 5%.Culturing process detects bacterium dense (OD600nm).When bacterium is dense while reaching 0.5-1, ventilating mode is made into from the ventilating mode of the logical pressurized air in top (surface ventilation) of fermentor tank, dissolved oxygen amount is controlled at 0-2%, preferably 0.1-1%, more excellent 0.2%.As the dense 2-2.5 that reaches of fermented liquid bacterium, supplement the glucose of 600ml 50%, continue to be cultured to the plateau of thalli growth, add the phenol deactivation thalline of final concentration 0.5%.Then enter the capsular polysaccharide extraction process stage.
2. the extraction of streptococcus pneumoniae 19F capsular polysaccharide
The nutrient solution of above-mentioned deactivation, adopts the centrifugal 30min of 15000g to remove thalline and bacterial chip, and supernatant liquor further adopts the mode of micro-filtration or Depth Filtration to remove remaining particulate matter.Solution after clarification adopts the ultrafiltration system of 100kDa to concentrate and remove the small molecules composition in nutrient solution, the final about 3L of concentrated solution that obtains.
Substep alcohol precipitation: above-mentioned concentrated solution slowly adds precooling dehydrated alcohol while stirring, and every 100ml concentrated solution adds 33.3ml dehydrated alcohol, fully mixes rear 4 ℃ of hold over night.The centrifugal 30min of 15000g removes precipitation, and supernatant continues slowly to add while stirring ethanol to final concentration 80%, fully mixes rear 4 ℃ of hold over night.The centrifugal 30min collecting precipitation of 15000g, precipitation adopts 0.2M NaCl solution 600ml to redissolve.The centrifugal 30min of 15000g removes and precipitates to obtain supernatant, is Crude polysaccharides solution.
Molecular sieve exclusion chromatography separates: above-mentioned supernatant adopts other molecular sieve exclusion chromatography fillers of molecular sieve exclusion chromatography Sepharose CL-4B(Sephacryl S-200, Sepharose 4 Fast Flow, Superdex 200 etc. also can adopt) separate, UV-detector (wavelength 206nm) and differential detector monitors sample peak, collect the sample peak of relative molecular weight kd≤0.20.Polysaccharide sample after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains refining polysaccharide, and refining polysaccharide yield is 90-120mg/L fermented liquid.Refining polysaccharide is stored in-20 ℃ of following refrigerators.The distribution situation of refining polysaccharide on chromatography column sees appendix two, and as can be seen from the figure, the in the situation that of having higher absorption peak under 206nm, absorption value under 280nm (mainly representing protein peak) is very low, and the visible polysaccharide extracting has higher purity.
3. refining polysaccharide characterized
(1) biochemical identification project and method are referring to European Pharmacopoeia 5.0;
(2) adopt
1h-NMR carries out structure characteristic analysis to refining polysaccharide;
(3) adopt Pn19F specific serum (purchased from serum institute of country of Denmark) to utilize immune double diffusion or reversible circulation immunoelectrophoresis to identify refining polysaccharide;
(4) adopt molecular sieve exclusion chromatography (Sepharose CL-4B or Sepharose CL-2B) to measure the relative molecular weight of refining polysaccharide.
Embodiment 2: the cultivation of streptococcus pneumoniae 9V and the extraction of Pn9V-Ps
1. the cultivation of streptococcus pneumoniae 9V
Bacterial classification is from American Type Culture Collecti (ATCC 6309).
The cellar culture method of streptococcus pneumoniae has been that this area is in common knowledge, and spendable liquid nutrient medium comprises: TSB, CY, BHI, Hoeprich ' s substratum and improved formulations thereof etc.
From-70 ℃ of refrigerators, get streptococcus pneumoniae 9V type work seed, be inoculated in containing on 10% defiber sheep blood nutrient agar panel 36 ℃ ± 1 ℃, 5%CO
2in incubator, cultivate 18-24h.Checking contamination-free.
Above-mentioned culture is scraped, is seeded in several 1L(substratum loading amount 500ml) in triangular flask, in 36 ℃ ± 2 ℃, 5%CO
2in incubator, cultivate 14-20h, in this process, monitor cell density (OD600nm).In the time that cell density reaches 2.0-2.5, adopt plate streaking and microscopy method validation contamination-free to occur.
Above-mentioned 1.5L culture is inoculated in 50L fermentor tank (loading amount 30L) to culture condition: 36 ℃ ± 2 ℃ of culture temperature, pH maintains 7.0 ± 0.2, rotating speed 80r/min.After inoculation, cultivate the initial stage to adopt the logical compressed-air actuated ventilating mode from fermentor tank bottom, dissolved oxygen amount is controlled at 1-10%, preferably 3-7%, more excellent 4%.Culturing process detects bacterium dense (OD600nm).When bacterium is dense while reaching 0.5-1, ventilating mode is made into from the ventilating mode of the logical pressurized air in top (surface ventilation) of fermentor tank, dissolved oxygen amount is controlled at 0-2%, preferably 0.05-1%, more excellent 0.1%.As the dense 2-2.5 that reaches of fermented liquid bacterium, supplement the glucose of 600ml 50%, continue to be cultured to the plateau of thalli growth, add the phenol deactivation thalline of final concentration 0.5%.Then enter the capsular polysaccharide extraction process stage.
2. the extraction of streptococcus pneumoniae 9V capsular polysaccharide
The nutrient solution of above-mentioned deactivation, adopts the centrifugal 30min of 15000g to remove thalline and bacterial chip, and supernatant liquor further adopts the mode of micro-filtration or Depth Filtration to remove remaining particulate matter.Solution after clarification adopts the ultrafiltration system of 100kDa to concentrate and remove the small molecules composition in nutrient solution, the final about 3L of concentrated solution that obtains.
Substep alcohol precipitation: above-mentioned concentrated solution slowly adds precooling dehydrated alcohol while stirring, and every 100ml concentrated solution adds 33.3ml dehydrated alcohol, fully mixes rear 4 ℃ of hold over night.The centrifugal 30min of 15000g removes precipitation, and supernatant continues slowly to add while stirring ethanol to final concentration 80%, fully mixes rear 4 ℃ of hold over night.The centrifugal 30min collecting precipitation of 15000g, precipitation adopts 0.2M NaCl solution 600ml to redissolve.The centrifugal 30min of 15000g removes and precipitates to obtain supernatant, is Crude polysaccharides solution.
Molecular sieve exclusion chromatography separates: above-mentioned supernatant adopts other molecular sieve exclusion chromatography fillers of molecular sieve exclusion chromatography Sepharose CL-2B(Sephacryl S-200, Sepharose 4 Fast Flow, Superdex 200 etc. also can adopt) separate, UV-detector (wavelength 206nm) and differential detector monitors sample peak, collect the sample peak of relative molecular weight kd≤0.45.Polysaccharide sample after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains refining polysaccharide, and refining polysaccharide yield is 100-130mg/L fermented liquid.Refining polysaccharide is stored in-20 ℃ of following refrigerators.The distribution situation of refining polysaccharide on chromatography column sees appendix three, and as can be seen from the figure, the in the situation that of having higher absorption peak under 206nm, absorption value under 280nm (mainly representing protein peak) is very low, and the visible polysaccharide extracting has higher purity.
3. refining polysaccharide characterized
Referring to the implementation method of this part content in embodiment mono-, polysaccharide average molecular flow measurement filler adopts Sepharose CL-2B.
Embodiment 3: the cultivation of streptococcus pneumoniae 5 types and the extraction of Pn5-Ps
1. the cultivation of streptococcus pneumoniae 5 types
Bacterial classification is from American Type Culture Collecti (ATCC 6305).
The cellar culture method of streptococcus pneumoniae has been that this area is in common knowledge, and spendable liquid nutrient medium comprises: TSB, CY, BHI, Hoeprich ' s substratum and improved formulations thereof etc.
From-70 ℃ of refrigerators, get streptococcus pneumoniae 5 type work seeds, be inoculated in containing on 10% defiber sheep blood nutrient agar panel 37 ℃ ± 2 ℃, 5%CO
2in incubator, cultivate 18-24h.Checking contamination-free.
Above-mentioned culture is scraped, is seeded in several 1L(substratum loading amount 500ml) in triangular flask, in 37 ℃ ± 1 ℃, 5%CO
2in incubator, cultivate 14-20h, in this process, monitor cell density (600nm).In the time that cell density reaches 2.0-2.5, adopt plate streaking and microscopy method validation contamination-free to occur.
Above-mentioned 1.5L culture is inoculated in 50L fermentor tank (loading amount 30L) to culture condition: 37 ℃ ± 2 ℃ of culture temperature, pH maintains 7.2 ± 0.2, rotating speed 100r/min.After inoculation, cultivate the initial stage to adopt the logical compressed-air actuated ventilating mode from fermentor tank bottom, dissolved oxygen amount is controlled at 1-10%, preferably 3-7%, more excellent 5%.Culturing process detects bacterium dense (OD600nm).When bacterium is dense while reaching 0.5-1, ventilating mode is made into from the ventilating mode of the logical pressurized air in top (surface ventilation) of fermentor tank, dissolved oxygen amount is controlled at 0-2%, preferably 0.1-1%, more excellent 0.2%.As the dense 2-2.5 that reaches of fermented liquid bacterium, supplement the glucose of 600ml 50%, continue to be cultured to the plateau of thalli growth, add the phenol deactivation thalline of final concentration 0.5%.Then enter the capsular polysaccharide extraction process stage.
2. the extraction of streptococcus pneumoniae CP5
The nutrient solution of above-mentioned deactivation, adds isopyknic water for injection to reduce the viscosity of fermented liquid, adopts afterwards the centrifugal 30min of 15000g to remove thalline and bacterial chip, and supernatant liquor further adopts the mode of micro-filtration or Depth Filtration to remove remaining particulate matter.Solution after clarification adopts the ultrafiltration system of 100kDa to concentrate and remove the small molecules composition in nutrient solution, the final about 3L of concentrated solution that obtains.
Substep alcohol precipitation: above-mentioned concentrated solution slowly adds precooling dehydrated alcohol while stirring, and every 100ml concentrated solution adds 25ml dehydrated alcohol, fully mixes rear 4 ℃ of hold over night.The centrifugal 30min of 15000g removes precipitation, and supernatant continues slowly to add while stirring ethanol to final concentration 80%, fully mixes rear 4 ℃ of hold over night.The centrifugal 30min collecting precipitation of 15000g, precipitation adopts 0.2M NaCl solution 600ml to redissolve.The centrifugal 30min of 15000g removes and precipitates to obtain supernatant, is Crude polysaccharides solution.
Molecular sieve exclusion chromatography separates: above-mentioned supernatant adopts other molecular sieve exclusion chromatography fillers of molecular sieve exclusion chromatography Sepharose CL-2B(Sephacryl S-200, Sepharose 4 Fast Flow, Superdex 200 etc. also can adopt) separate, UV-detector (wavelength 206nm) and differential detector monitors sample peak, collect the sample peak of relative molecular weight kd≤0.60.Polysaccharide sample after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains refining polysaccharide, and refining polysaccharide yield is 70-100mg/L fermented liquid.Refining polysaccharide is stored in-20 ℃ of following refrigerators.The distribution situation of refining polysaccharide on chromatography column sees appendix four, and as can be seen from the figure, the in the situation that of having higher absorption peak under 206nm, absorption value under 280nm (mainly representing protein peak) is very low, and the visible polysaccharide extracting has higher purity.
3. refining polysaccharide characterized
Referring to the implementation method of this part content in embodiment mono-, polysaccharide average molecular flow measurement filler adopts Sepharose CL-4B.
Embodiment 4,6B type streptococcus pneumoniae
The cultivation of streptococcus pneumoniae, method is as follows:
After stirred-tank fermenter inoculation, pH is controlled at 6.5, rotating speed 50r/min, cultivates initial stage employing from the logical pressurized gas in fermentor tank bottom, and dissolved oxygen amount is controlled at 1%, in the time that fermented liquid bacterium dense (OD600nm) reaches 0.5, changing ventilating mode is logical pressurized gas from the top of fermentor tank, and dissolved oxygen is controlled at 0.01%, when fermented liquid bacterium is dense while reaching 2, appropriate supplementary carbon source, continues to be cultured to deactivation thalline after the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove the small molecules composition in thalline, bacterial chip and nutrient solution, obtain concentrated solution; After b, concentrated solution slowly add ethanol to final concentration to be 15%, 2 ℃ of hold over night; C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 60%, and 2 ℃ of hold over night; D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution;
Refining of streptococcus pneumoniae pod membrane Crude polysaccharides, method is as follows:
Gained raw sugar solution, adopts Superdex 200 molecular sieve exclusion chromatographies to separate, and the standard according to the pneumonia polysaccharide of each type in European Pharmacopoeia 5.0 editions is collected macromolecular polysaccharide; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
The cultivation of streptococcus pneumoniae, method is as follows:
After stirred-tank fermenter inoculation, pH is controlled at 8.0, rotating speed 200r/min, cultivates initial stage employing from the logical pressurized gas in fermentor tank bottom, and dissolved oxygen amount is controlled at 10%, in the time that fermented liquid bacterium dense (OD600nm) reaches 1, changing ventilating mode is logical pressurized gas from the top of fermentor tank, and dissolved oxygen is controlled at 2%, when fermented liquid bacterium is dense while reaching 2.5, appropriate supplementary carbon source, continues to be cultured to deactivation thalline after the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove the small molecules composition in thalline, bacterial chip and nutrient solution, obtain concentrated solution; After b, concentrated solution slowly add ethanol to final concentration to be 35%, 8 ℃ of hold over night; C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 90%, and 8 ℃ of hold over night; D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution;
Refining of streptococcus pneumoniae pod membrane Crude polysaccharides, method is as follows:
Gained raw sugar solution, adopts Superdex 200 molecular sieve exclusion chromatographies to separate, and the standard according to the pneumonia polysaccharide of each type in European Pharmacopoeia 5.0 editions is collected macromolecular polysaccharide; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
The cultivation of streptococcus pneumoniae, method is as follows:
After stirred-tank fermenter inoculation, pH is controlled at 7, rotating speed 100r/min, cultivates initial stage employing from the logical pressurized gas in fermentor tank bottom, and dissolved oxygen amount is controlled at 5%, in the time that fermented liquid bacterium dense (OD600nm) reaches 0.75, changing ventilating mode is logical pressurized gas from the top of fermentor tank, and dissolved oxygen is controlled at 1%, when fermented liquid bacterium is dense while reaching 2.1, appropriate supplementary carbon source, continues to be cultured to deactivation thalline after the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove the small molecules composition of particulate matter in thalline, bacterial chip, nutrient solution, obtain concentrated solution; After b, concentrated solution slowly add ethanol to final concentration to be 20%, 5 ℃ of hold over night; C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 75%, and 5 ℃ of hold over night; D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution;
Refining of streptococcus pneumoniae pod membrane Crude polysaccharides, method is as follows:
Gained raw sugar solution, adopts Sepharose 4 Fast Flow molecular sieve exclusion chromatographies to separate, and the standard according to the pneumonia polysaccharide of each type in European Pharmacopoeia 5.0 editions is collected macromolecular polysaccharide; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
The cultivation of streptococcus pneumoniae, method is as follows:
After stirred-tank fermenter inoculation, pH is controlled at 7.5, rotating speed 150r/min, cultivates initial stage employing from the logical pressurized gas in fermentor tank bottom, and dissolved oxygen amount is controlled at 8%, in the time that fermented liquid bacterium dense (OD600nm) reaches 0.6, changing ventilating mode is logical pressurized gas from the top of fermentor tank, and dissolved oxygen is controlled at 0.05%, when fermented liquid bacterium is dense while reaching 2.2, appropriate supplementary carbon source, continues to be cultured to deactivation thalline after the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove the small molecules composition of particulate matter in thalline, bacterial chip, nutrient solution, obtain concentrated solution; After b, concentrated solution slowly add ethanol to final concentration to be 30%, 8 ℃ of hold over night; C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 85%, and 8 ℃ of hold over night; D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution;
Refining of streptococcus pneumoniae pod membrane Crude polysaccharides, method is as follows:
Gained raw sugar solution, adopts Sephacryl S-200 molecular sieve exclusion chromatography to separate, and collects macromolecular polysaccharide according to the pneumonia polysaccharide of each type in the standard in European Pharmacopoeia 5.0 editions; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
The cultivation of streptococcus pneumoniae, method is as follows:
After stirred-tank fermenter inoculation, PH is controlled at 7, rotating speed 70r/min, rotating speed is cultivated initial stage employing from the logical pressurized gas in fermentor tank bottom, and dissolved oxygen amount is controlled at 3%, when fermented liquid bacterium is dense while reaching 0.5, changing ventilating mode is logical pressurized gas from the top of fermentor tank, and dissolved oxygen is controlled at 0.1%, when fermented liquid bacterium is dense while reaching 2, appropriate supplementary carbon source, continues to be cultured to deactivation thalline after the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove the small molecules composition in thalline, bacterial chip and nutrient solution, obtain concentrated solution; After b, concentrated solution slowly add ethanol to final concentration to be 15%, 2 ℃ of hold over night; C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 60%, and 2 ℃ of hold over night; D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution;
Refining of streptococcus pneumoniae pod membrane Crude polysaccharides, method is as follows:
Gained raw sugar solution, adopts Sepharose CL-4B molecular sieve exclusion chromatography to separate, and collects macromolecular polysaccharide according to the pneumonia polysaccharide of each type in the standard in European Pharmacopoeia 5.0 editions; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
The cultivation of streptococcus pneumoniae, method is as follows:
After stirred-tank fermenter inoculation, PH is controlled at 7.5, rotating speed 150r/min, rotating speed is cultivated initial stage employing from the logical pressurized gas in fermentor tank bottom, and dissolved oxygen amount is controlled at 7%, when fermented liquid bacterium is dense while reaching 1, changing ventilating mode is logical pressurized gas from the top of fermentor tank, and dissolved oxygen is controlled at 1.0%, when fermented liquid bacterium is dense while reaching 2.5, appropriate supplementary carbon source, continues to be cultured to deactivation thalline after the plateau of thalli growth.
The extraction of streptococcus pneumoniae capsular polysaccharide, method is as follows:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove the small molecules composition in thalline, bacterial chip and nutrient solution, obtain concentrated solution; After b, concentrated solution slowly add ethanol to final concentration to be 35%, 8 ℃ of hold over night; C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 90%, and 8 ℃ of hold over night; D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution;
Refining of streptococcus pneumoniae pod membrane Crude polysaccharides, method is as follows:
Gained raw sugar solution, adopts Sepharose CL-2B molecular sieve exclusion chromatography to separate, and collects macromolecular polysaccharide according to the pneumonia polysaccharide of each type in the standard in European Pharmacopoeia 5.0 editions; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
The preparation of the streptococcus pneumoniae capsular polysaccharide of other types as: 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F type, preparation method is identical with embodiment 1.
Claims (9)
1. a preparation method for streptococcus pneumoniae capsular polysaccharide, the method comprises the following steps, the cultivation of streptococcus pneumoniae, deactivation, the extraction of streptococcus pneumoniae pod membrane Crude polysaccharides, refining of streptococcus pneumoniae pod membrane Crude polysaccharides, is characterized in that,
The extraction of described streptococcus pneumoniae pod membrane Crude polysaccharides, method is as follows:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove the small molecules composition in thalline, bacterial chip and nutrient solution, obtain concentrated solution; After b, concentrated solution slowly add ethanol to final concentration to be 15-35%, 2-8 ℃ of hold over night; C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 60-90%, and 2-8 ℃ of hold over night; D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution;
Wherein, described streptococcus pneumoniae pod membrane Crude polysaccharides refining, method is as follows:
Gained raw sugar solution, adopts molecular sieve exclusion chromatography to separate, and collects macromolecular polysaccharide according to the pneumonia polysaccharide of each type in the standard in European Pharmacopoeia 5.0 editions; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
2. preparation method as described in claim 1, is characterized in that, wherein, the extraction of described streptococcus pneumoniae pod membrane Crude polysaccharides, step is as follows:
A, get the culture of streptococcus pneumonia liquid after deactivation, remove particulate matter and small molecules composition in thalline, bacterial chip, nutrient solution, obtain concentrated solution; After b, concentrated solution slowly add ethanol to final concentration to be 20-30%, 2-8 ℃ of hold over night; C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 75-85%, and 2-8 ℃ of hold over night; D, gained leave standstill liquid centrifugal collecting precipitation, after precipitation water or NaCl solution redissolve, and centrifugal removal precipitation, gained supernatant is raw sugar solution.
3. preparation method as described in claim 1, is characterized in that, wherein, the extraction of described streptococcus pneumoniae pod membrane Crude polysaccharides, step is as follows:
A, get culture of streptococcus pneumonia liquid after the thalline of deactivation, adopt the centrifugal 30min of 15000g to remove thalline and bacterial chip, supernatant liquor further adopts the mode of micro-filtration or Depth Filtration to remove remaining particulate matter; Solution after clarification adopts the ultrafiltration system of 100kDa to concentrate and remove the small molecules composition in nutrient solution, the final concentrated solution that obtains; After b, concentrated solution slowly add ethanol to final concentration to be 25%, 2-8 ℃ of hold over night; C, gained leave standstill the centrifugal removal precipitation of liquid, after supernatant continues to add ethanol to final concentration and is 80%, and 2-8 ℃ of hold over night; D, gained leave standstill the centrifugal 30min collecting precipitation of liquid 15000g, and precipitation adopts 0.2M NaCl solution to redissolve, redissolution liquid 15000g, and centrifugal 30min removes and precipitates to obtain supernatant, is Crude polysaccharides solution.
4. preparation method as described in claim 1, wherein, the refining employing molecular sieve exclusion chromatography of described streptococcus pneumoniae pod membrane Crude polysaccharides, it is characterized in that, molecular sieve exclusion chromatography filler used is optional: Sepharose CL-2B, Sepharose CL-4B, Sephacryl S-200, Sepharose 4 Fast Flow or Superdex 200; Standard according to the pneumonia polysaccharide of each type in European Pharmacopoeia 5.0 editions is collected macromolecular polysaccharide; Polysaccharide after separation adopts the desalination of 30kDa ultra-filtration membrane bag, then after lyophilize, obtains streptococcus pneumoniae capsular polysaccharide.
5. preparation method as claimed in claim 1, it is characterized in that, wherein, the cultivation of described streptococcus pneumoniae, step is as follows: after stirred-tank fermenter inoculation, pH is controlled at 6.5-8.0, rotating speed 50-200r/min, cultivate initial stage employing from the logical pressurized gas in fermentor tank bottom, dissolved oxygen amount is controlled at 1-10%, when fermented liquid bacterium is dense while reaching 0.5-1, changing ventilating mode is the logical pressurized gas in top from fermentor tank, dissolved oxygen is controlled at 0.01-2%, when fermented liquid bacterium is dense while reaching 2-2.5, appropriate supplementary carbon source, continue to be cultured to deactivation thalline after the plateau of thalli growth.
6. preparation method as claimed in claim 5, it is characterized in that, wherein, the cultivation of described streptococcus pneumoniae, step is as follows: after stirred-tank fermenter inoculation, PH is controlled at 7-7.5, rotating speed 70-150r/min, cultivate initial stage employing from the logical pressurized gas in fermentor tank bottom, dissolved oxygen amount is controlled at 3-7%, when fermented liquid bacterium is dense while reaching 0.5-1, changing ventilating mode is the logical pressurized gas in top from fermentor tank, dissolved oxygen is controlled at 0.1-1.0%, when fermented liquid bacterium is dense while reaching 2-2.5, appropriate supplementary carbon source, continue to be cultured to deactivation thalline after the plateau of thalli growth.
7. preparation method as claimed in claim 5, it is characterized in that, the cultivation of described streptococcus pneumoniae, step is as follows: after stirred-tank fermenter inoculation, 37 ℃ ± 2 ℃ of culture temperature, pH maintains 7.2 ± 0.2, rotating speed 100r/min, rotating speed is cultivated initial stage employing from the logical pressurized gas in fermentor tank bottom, dissolved oxygen amount is controlled at 5%, when fermented liquid bacterium is dense while reaching 0.5-1, changing ventilating mode is the logical pressurized gas in top from fermentor tank, dissolved oxygen is controlled at 0.2%, when fermented liquid bacterium is dense while reaching 2-2.5, appropriate supplementary carbon source, continue to be cultured to deactivation thalline after the plateau of thalli growth.
8. preparation method as claimed in claim 5, it is characterized in that, the applicable bacterial type of described cultural method comprises 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F type.
9. preparation method as claimed in claim 5, is characterized in that, is aseptic pressurized air to the gas passing in fermentor tank, or the composition gas of oxygen and nitrogen or carbonic acid gas.
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| CN107058421A (en) * | 2017-05-15 | 2017-08-18 | 中国农业科学院兰州畜牧与兽药研究所 | The method for extraction and purification of capsular polysaccharide in a kind of 336 type staphylococcus aureus |
| CN107058421B (en) * | 2017-05-15 | 2021-01-26 | 中国农业科学院兰州畜牧与兽药研究所 | Method for extracting and purifying capsular polysaccharide in 336 type staphylococcus aureus |
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